Treatment of fungal and/or protist infections

ABSTRACT

The present invention relates to the use of a polypeptides, comprising repeats of a peptide derived from a Heparan Sulphate Proteoglycan (HSPG) receptor binding region of an apolipoprotein, for treating or preventing a fungal and/or protist infection. The invention further relates to the use of such peptides for treating or preventing the contamination of surfaces or objects with such peptides.

The present invention relates to polypeptides, derivatives or analoguesthereof, with antimicrobial activity, and to nucleic acids encoding thesame. More specifically, the invention relates to polypeptides,derivatives or analogues thereof with antifungal and/or antiprotistactivity. The invention further provides the use of such polypeptides,derivatives, analogues or nucleic acids as medicaments, and also inmethods of treatment. The invention further extends to objects andsurfaces coated with the polypeptides.

Antimicrobial peptides are a key component of the innate immune system,generally containing 20-40 amino acids, having a net positive charge,and with the majority having been identified so far in non-mammalianspecies. Both of these factors limit their usefulness as therapeutics inhumans or mammals. This is due to difficulties in commercial productionof such large peptides, and the risk of adverse effects from peptides ofnon-human origin. By 2006, of the around 890 sequences listed in theTrieste international antimicrobial peptide database(http://www.bccm.units.it/˜tossi/amsdb.html), only 35 were of humanorigin, and of these only 3 are less than 20 amino acids in length. Someshort synthetic antimicrobial peptides have also been developed.However, these have the disadvantage of associated risks of antigenic ortoxic effects due to their non-human origin.

Such peptides have been characterised into six groups (Bradshaw, J. P.,Biodrugs, 2003: 17: 235-240), with the following three classes beingmost studied (Bowman H. G., Journal of internal Medicine, 2003:254:197-215):

-   -   (i) Linear peptides lacking cysteines and often with an        α-helical amiphipathic structure in solution, for example,        Hurman LL-37 (SEQ ID No 1):—LLGDFFRKSKEKIGKEFKRI VQRIKDFLRN        LYPRTES;    -   (ii) Peptides with 3 disulphide bonds, giving peptides vilh a        flat dimeric beta-sheet, for example, Hunan α-defensin:—HNP-1        (SEQ ID No.2)

-   -   (iii) Peptides with unusual bias in certain amino acids such as        proline, arginine, tryptophan or histidine, fat example, Pig        PR-39 (SEQ ID No.3):—RRRPRPPYLP RPRPPPFFPP RLPPRIPPGF PPRFPPRFP;        or cow indolicidin (SEQ ID No.4):—ILPWKWPWWP WRR.

Some peptides have been discovered that have the capacity to inhibit thegrowth of fungi. Examples of such peptides include Cecropins, Buforins,Pleuoridin Pyrrhocoricin, metalnikowin, sheperins, AcAMP1 and Ac-AMP2,Histatins, Tachyplesin II, Androctonin, Protegrin 1, α Defensins and βDefensins, Penaeidins, Tachycitin, Heliomicin, defensin protein WT1,alfAFP defensin So-d1-7, DmAMP1. Furthermore, some peptides have beenfound to additionally inhibit protozoa (for example Megainin, anddermaseptin). Others have been shown to inhibit both fungi and protozoa(for example Gambicin). However, at present, the mechanism of suchagents to impart their antifungal and/or antiprotozoan activities is notfully understood.

A number of antibacterial peptides that have been described in thescientific literature have strong cationic character, and often consistof arginine and lysine residues. However, not all peptides containingarghiine and lysine have antimicrobial activity. For example, Azama etal. (Peptides, 21: 327-330 (2000)) have reported that peptidederivatives of apolipoprotein E have a strong antibacterial action,comparable to that of gentamicin. However, Azuma's peptide apoE₁₃₄₋₁₅₁(18 amino acids in length) had no activity at all despite containingarginines both at positions 142 and 147. Similarly, Azuma demonstratedthat the peptide apoE₁₃₄₋₁₅₅ (22 amino acids in length) had very lowantibacterial activity, and the peptide apoE₁₃₄₋₁₅₉ (26 amino acids inlength) had greatly reduced antibacterial activity. Finally, Azuma etal. only investigated the antibacterial activity of the apoE derivedpeptides, and did not evaluate any other anti-microbial effects, forexample, activity against fungi or protists.

Following on from the research carried out by Azuma et. al., theinventor of the present invention investigated the action of certainpolypeptides based on apolipoproteins B and E, against viruses. Theinvestor established that certain polypeptides do have antiviralactivity. The results of his research are described in PCT/GB2004/005438and PCT/GB2004/005360. These antiviral polypeptides comprise tandemrepeats, and variants thereof, of the peptides: apoE₁₄₁₋₁₄₉(LRKLRKRLL—SEQ ID No.5) and apoB₃₃₅₉₋₃₃₆₇(RLTRKRGLK—SEQ ID No.6)as-well-as repeats of closely related modifications of SEQ ID No.5 orSEQ ID No.6. These peptides are either derived from or comprise the LDLreceptor/HSPG receptor binding region of apolipoproteins E and B. Whilethe mvrntor does not wish to be bound by any hypothesis, he considers itlikely that these antiviral polypeptides exert their antiviral actionsby a number of mechanisms, with those affecting viral attachment beingparticularly favoured. The inventor suggests that dimerlsation ofpeptides derived from the LDL receptor/HSPG receptor binding region ofthese apolipoproteins (as a tandem repeat or variant thereof) isimportant for an antiviral effect.

Despite the fact that antiviral agents are unrelated to antibacterialagents due to their different modes of action, on viruses and bacteria,respectively, the inventor also decided to investigate whetherpolypeptides, based on the antiviral peptides discussed above, also hadany antibacterial properties. The results of this research are describedin PCT/GB2005/000769. To his surprise, the inventor found that inaddition to antiviral activity, these polypeptides also exhibitedantibacterial activity.

The inventor of the present invention continued his investigations onthese polypeptides based on apolipoproteins B and E. Accordingly,despite the fact that antiviral agents, and antibacterial agents areunrelated to antifungal agents and agents exhibiting activity to othermicro-organisms, due to their different modes of action on viruses,bacteria, and fungi respectively, the inventor of the present inventionalso decided to investigate whether polypeptides, based on theantiviral/antibacterial peptides discussed above, had any anti-fungalproperties, or exhibited any activity against any other micro-organisms.

Specifically the inventor wondered whether construction of a repeat(e.g. tandem repeats) of peptides derived from the LDL receptor/HSPGreceptor binding region of these apoiipoprotems may have otherantimicrobial effects, for example, against fungi or othermicro-organisms, such as protozoa. In particular, the inventor wonderedif repeats of the apoE₁₄₁₋₁₄₉ region aright unexpectedly exhibit eitherantifungal and/or antiprotist activity. To his surprise, he found thatpolypeptides, as defined below, do exhibit antifungal activity, and alsoactivity against other micro-organisms, in addition to the antiviral andantibacterial activities, which had been demonstrated previously.

According to a first aspect of the present invention, there is provideduse of a porypeptide, or a derivative or analogue thereof, comprisingrepeats of a peptide derived from a Heparan Sulphate Proteoglycan (HSPG)receptor binding region of an apolipoprotein for the manufacture of amedicament for the treatment of a fungal and/or protist infection.

By the term “derivative or analogue thereof”, we mean a polypeptidewithin which amino acids residues are replaced by residues (whethernatural amino acids, non-natural amino acids or amino acid mimics) withsimilar side chains or peptide backbone properties. Additionally, eitherone or both terminals of such peptides may be protected by N andC-terminal protecting groups, for example, groups with similarproperties to acetyl or amide groups. It will be appreciated that theamino acid sequence may be varied, truncated or modified once the finalpolypeptide is termed or during the development of the repeated peptides(e.g. the 9-mer).

Preferably, the polypeptide of the invention comprises at least tworepeats of a peptide derived from an HSPG receptor binding region of anapolipoprotein. It will be appreciated that the polypeptide may compriserepeats of the same peptide (i.e. a homodimer or polymer of the samepeptide). Alternatively, the polypeptide may comprise a repeat of two ormore related peptides (i.e. a heterodimer or a polymer comprising two ormore peptide types of peptide monomer). If the polypeptide comprisesdifferent peptides, it will be appreciated that such peptides will sharethe characteristics that they are, or are derived from, an HSPG receptorbinding region of an apolipoprotein, as defined in the first aspect ofthe invention.

It is preferred that polypeptides according to the invention comprisedimers or polymers of such peptides linked N terminal to C terminal in afashion that would be known to one skilled in the art as a tandemrepeat. Accordingly, unless the context dictates otherwise, when werefer to “tandem repeats” herein, we mean a repeat of peptides that are,or are derived from, an HSPG receptor binding region of anapolipoprotein. Such tandem repeats may be homodimers (or polymers of asingle peptide) or may comprise a heterodimer (or polymer of relatedpeptides) as discussed above.

The term “peptides derived from” as used herein is intended to describeor include peptides from the HSPG receptor binding region of anapolipoprotein that have been modified. Suitable modification mayinclude amino acid subtitution, addition or deletion. The derivativepeptide or modified peptide is arranged as a tandem repeat in accordancewith the first aspect of the invention. Surprisingly, and completelycontrary to expectation, polypeptides, derivatives or analogues thereofaccording to the first aspect of the invention have been shown toexhibit antifungal and/or antiprotist activity.

When the term “a truncation thereof” is used herein, we mean that thepolypeptide according to the invention or the constituent peptide isreduced in size by removal or deletion of one or more amino acids. Thereduction of amino acids may be by removal of residues from the C or Nterminal of the polypeptide, or may be by deletion of one or more aminoacids from within the constituent peptides.

The inventors have previously found that polypeptides as defined abovehave antiviral and antibacterial activity. However, to the inventors'surprise, when the polypeptides according to the first aspect weretested on fungi and protists, they also showed antifungal efficacy andalso activity against protists, as shown in the Examples. Hence, it isthe inventors' belief that they have therefnre shown a new medicalindication for these polypeptides.

The medicament according to the first aspect of the invention may beused in the medical treatment of banians or for veterinary use. Themedicament is preferably used to treat fungal infections of humans andother mammals.

The polypeptide used to prevent or treat fungal and/or protistinfections is preferably derived from the same species as the subjectbeing treated. When that subject is a human it is preferred that thepolypeptide is based on repeats derived from human apolipoproteins.

By the term “fungal infection”, we mean an infection with, or caused by,a fungus. Infectious that may be treated include: blastomycosis,coccidiodomycosis, cryptococcosis, histoplasmosis, sporotrichosis,chromoblastomycosis, lobomycosis, dermoatophytosis, dermatomycosis,onychomycosis, piedra, mycetoma, fusariosis, pityriasis versicolor,tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea favosa,tinea nigra, tinea pedis, phaeohyphomycosis, rhinosporidiosis,aspergillosis, mycotic keratitis, candidiasis.

By the term “protist infection”, we mean an infection with, or causedby, a protist. Diseases caused by infection with Protista includegiardiasis and other gastrointestinal disorders including amoebicdysentery and diarrhoea, cutaneous and visceral leishmaniasis, Chagas'disease, coccidiosis, ick, trichomoniasis, African sleeping sickness,red tides, toxoplasmosis, malaria, and microbial keratitis, includingAcanthamoeba keratitis.

In general, antiviral agents, such as acyclovir, ribavirin, orenmvirtide (T-20), are rarely useful against antifungal infections dueto their completely different modes of action. Similarly, antiihngalagents, such as Butenafine, Botoconazole, or Naftifine, are rarelyuseful against viral infections. Accordingly, the inventor of thepresent invention, was very surprised that the polypeptides according tothe first aspect of the invention showed antiviral, and antibacterialefficacy, and also activity against fungal and/or protist infections. Itwas completely unexpected that the peptides according to the inventionwould have activity across kingdoms, i.e., activity in the KingdomMonera (bacteria and viruses), and the Kingdom Fungi, and also theKingdom Protista. The inventor was surprised that the peptides accordingto the invention had such versatility and such a broad range ofactivity, as this very rarely occurs.

Whilst the inventor does not wish to be bound by any hypothesis, he hassuggested that the antifungal/antiprotist mechanism of action by thepolypeptides in accordance with the invention, may involve a directdamaging effect to the fungus or protist, either mediated through themembrane, or through targeting a site within the fungus or psotist. Inaddition, blockade of attachment or entry of intracellular parasites(e.g. Plasmodium) into host cells is also possible. It is possibly forthese reasons that only a surprisingly small number of peptide sequences(i.e. those defined by the first aspect of the invention) have beenfound to be effective against fungi and/or protists.

The majority of the polypeptides according to the invention weresurprisingly active as both antifungal and/or antiprotist agents, andalso antiviral agents and antibacterial agents. It is possible that insome embodiments of the invention, the peptides may exhibit combinationsof the above antimicrobial activities. For example, the peptides mayexhibit either aiirifhngal or antiprotist activity (and variouscombinations of antibacterial and antiviral activity). Preferredpolypeptides according to the first aspect exhibit antifungal andantiprotist activity and in addition antibacterial activity and/orantiviral activity. It will be apparent that this quadruple activityexhibited by the polypeptides is most advantageous, as they may be usedto prevent or treat fungal infections, protist infections and also viraland bacterial infections, preferabiy, simultaneously.

The polypeptides according to the first aspect of the invention maycomprise repeats of peptides derived from a Heparan SulphateProteoglycan (HSPG) receptor binding region of human apolipoprotein B orhuman apolipoprotein E. It is preferred that the polypeptide accordingto the first aspect of the invention comprises a tandem repeat (asdefined above) of peptides derived from an apolipoprotein B LDL receptorbinding domain cluster B, as defined by Law and Scott (J. Lipid Res,1990, 31:1109-20), or alternatively, from an apolipoprotein E LDLreceptor binding domain cluster B (J. Lipid. Res. 1995, 36:1905-1918).The apolipoprotein B LDL receptor binding domain chaster B may belocated within an HSPG receptor binding region of apolipoprotein B, andthe apolipoprotein E LDL receptor binding domain cluster B ofapolipoprotein E may be located within an HSPG binding domain ofapolipoprotein E.

The inventor conducted exhaustive experiments to assess the antifungaland antiprotist activity of peptides from apolipoproteins andderivatives thereof. Peptides and derivatives from ApoE and ApoB were aparticular focus. The inventor found that the apoE₁₄₁₋₁₄₉ monomericsequence (see Table 1 and SEQ ID No.5): the apoB₃₃₅₉₋₃₃₆₇ (see Table 1and SEQ ID No. 6) and the modified apoB₃₃₅₉₋₃₃₆₇ (see Table 1 and SEQ IDNo.7) had no detectable antifungal activity. However, surprisingly, theinventor found that repeats of such peptides (i.e. polypeptides inaccordance with the first aspect of the present invention), do exhibitantifungal and/or antiprotist activity. Examples 1 and 4 illustrate theantifungal efficacy of the polypeptides according to the invention, andExamples 2 and 5 illnstrate the antiprotisi activity of the polypeptidesaccording to the invention.

While the inventor does not wish to be bound by any hypothesis, theinventor believes that the cationic amino acid residues in theapoE₁₄₁₋₁₄₉ peptides (based on SEQ ID No.5) and apoB₃₃₅₉₋₃₃₆₇ peptides(based on SEQ ID. No.6) and modified apoB₃₃₅₉₋₃₃₆₇ peptides (based onSEQ ID No.7) when in the form of tandem repeats allows antifungalactivity and antiprotist activity to occur. The inventor has alsoestablished that certain derivatives of these peptides also haveantifungal and/or antiprotist activity, including modifications andtruncations of the peptide sequences.

The inventor carried out some detailed analysis of polypeptides withantifungal and ardiprotist activity and in particular those based onrepeats of peptides derived from the Heparan Sulphate Proteoglycan(HSFG) receptor binding region of apolipoprotein B or apolipoprotein E.The inventor produced a sequence alignment between the amino acids ofapoE₁₄₁₋₁₄₉ (i.e. the 9-mer of SEQ ID No.5), aligned with the aminoacids of apoB₃₃₅₉₋₃₃₆₇ (i.e. the 9-mer of SEQ ID No. 6), and also themodified form of apoB₃₃₅₉₋₃₃₆₇ (i.e. the 9-mer of SEQ ID No.7). Thesequence alignment is shewn in Table 1. It will be appreciated thatthese three 9-mers, or derivatives thereof, are repeated in thepolypeptides according to the present invention to form at least an18-mer, which may be optionally truncated.

TABLE 1 Analysis of effective peptide sequences exhibitingantifungal/antiprotist properties 0 1 2 3 4 5 6 7 8 9 L R K L R K R L L— apoE (141-149) - SEQ ID No. 5 — R L T R K R G L K apoB (3359-3367) -SEQ ID No. 6 — L R T R K R G R K Modified apoB (3359-3367) - SEQ ID No.7 Indicates residue is the same residues in apoB 3359-3367 and apoE(141-149)

In the light of this alignment data, the inventor noticed that there wasa recurring (conserved) amino acid motif in each of the antifungalpolypeptides comprising tandem repeats of a peptide derived from aHeparan Sulphate Proteoglycan (HSFG) receptor binding region ofapoiipoprotein B (apoB₃₃₅₉₋₃₃₆₇ (SEQ ID No.6)), or the modifiedapolipoprotein B (apoB₃₃₅₉₋₃₃₆₇ (SEQ ID No.7)), or apolipoprotein E(apoE₁₄₁₋₁₄₉ (SEQ ID No.5)), or a truncation thereof. This motifcorresponds to a tripeptide: Arginine-Lysine-Arginine (RKR), which isfound at amino acid residues designated: 4,5,6 of SEQ ID No.5, and SEQID No.7, and SEQ ID No.6. The inventor noticed that all of thepolypeptides according to the invention exhibiting antifungal and/orantiprotist activity comprise these RKR motifs.

Therefore, it is especially preferred that the polypeptide according tothe invention comprises at least two RKR motifs (i.e. the polypeptidecomprises a tandem repeat of peptides comprising RKR motifs).

It will be appreciated that polypeptides according to the presentinvention comprise at least two or more RKR motifs (i.e. one RKR motifper repeat). In situations where the polypeptide comprises a trimer (3×)repeat, or tetranicr (4×) repeat, or an even greater number of repealthe polypeptide preferably comprises at least three, or at least fourRKR motifs, respectively.

Preferred peptides comprise two RKR motifs and consist of 18 amino acidor less.

In one embodiment of the invention, the polypeptide according to thefirst aspect may comprise a dtiner repeat of the peptide comprising theRKR motif and preferably has formula I:

{abcRKRxyz}+{a′b′c′RKRx′y′z′}

wherein a, b, c, a′, b′, c′, x, y, z, x′, y′, z′ are amino acidresidues, and wherein the polypeptide comprise peptide abcRKRxyz andpeptide a′b′c′RKRx′y′z′ which are repents of SEQ ID No.5, SEQ ID No.6,or SEQ ID No.7 and derivatives thereof. Such derivatives may compriseSEQ ID No.5, SEQ ID No.6, or SEQ ID No7, wherein at least one amino acidresidue of that peptide, other than the RKR motifs, may be replaced byan Arginioe (R), Tyrosine (Y), Methionine (M), Isoleucine (I),Phenylalanine (F), Tryptophan (W), Cysteine (C) or a derivative thereof.The peptide may also comprise a Histidine (H) substitution.

It is preferred that amino acid substitutions are with a Arginine (R),Phenylalanine (F) or Tryptophan (W) residue and most preferably, aTryptophan (W) residue, or a derivative thereof

Suitably, one or more, more suitably, two or more, and even moresuitably, three or more amino acid residues may be replaced by anArginine (R), Tyrosine (Y), Methionine (M), Isoleucine (I),Phenylalanine (F), Tryptophan (W), Cysteine (C) or derivative thereof.In one embodiment of the invention it is preferred that four or more,more preferably, five or more, and even, more preferably, six or moreamino acid residues of the polypeptide according to the first aspect ofthe invention may be replaced by these amino acids or a derivativethereof. Preferably, the replaced or substituted residue is the first,second, third, seventh, eighth, ninth, tenth, eleventh, twelfth,sixteenth, seventeenth, or eighteenth residue of the peptide defined byformula I.

The polypeptide according to the invention may comprise 18 amino acids(or derivatives thereof), and thereby correspond to the sequence definedby formula I with or without the substitutions discussed above. In thiscase, amino acid position 1 corresponds to a; position 2 corresponds tob; position 3 corresponds to c, position 4 corresponds to the amino aridR (of the RKR motif), and so on.

However, the inventor has surprisingly found that truncated polypeptidesbased on formula I also have efficacy as antifungal agents, and/orantiprotist agents. Accordingly, preferred polypeptides or derivativesthereof may have less than 18 amino acids. For instance, somepolypeptides according to the first aspect of the invention may be 17,16, 15, 14, 13, 12, 11, 10 or less amino acids in length. Deletions arepreferably made at positions 1, 2, 8, 9, 10, 11, 17 and/or 18 of thepolypeptide defined by formula I.

The inventor has also surprisingly found that polypeptides based onformula I but having additional amino acid residues, also have efficacyas antifungal/antiprotist agents. Accordingly, preferred polypeptides orderivatives thereof may have greater than 18 amino acids. For instance,some polypeptides according to the first aspect of the invention may be19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more amino acids inlength. Additions may be made to the N or C-termmal or in the core ofthe polypeptide. Additions may be made either before residue ‘a’ (i.e.at the N-terminal end of the polypeptide), or before ‘a′’ (i.e. in thecore of the polypeptide), as defined, in formula 1. Additions may bemade either after residue ‘z’ (i.e. in the core of the peptide) or after‘z′’ (at the C-terminal end of the peptide), as defined in formula I.

However, the addition is preferably made at position 0, 1, 2, 8, 9, 10,11, 17 and/or 18 of the peptide defined by formula I. Most preferably,additions are made before position 0 of the peptide; i.e. amino acidsare added to the N-terminal before the first amino acid at residue ‘a’defined by formula I.

The polypeptide according to formula I may preferably comprise thefollowing amino acids:

-   a & a′=is independently selected from Arginine (R); Tyrosine (Y):    Methionine (M); Isoleucine (I); Phenylalanine (F): Tryptophan (W);    Leucine (L); Lysine (K); Histidine (H); Cysteine (C); or is deleted;-   b & b′=is independently selected from Arginine (R); Tyrosine (Y);    Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W);    Leucine (L); Lysine (K); Cysteine (C); or is deleted;-   c & c′=is independendy selected from Arginine (R); Tyrosine (Y);    Methionine (M); Isoluecine (I); Phenylalanine (F); Tryptophan (W);    Leucine (L); Lysine (K); Histidine (H); Cysteine (C) Threonine (T);    or is deleted;-   x & x′=is independently selected irom Arginine (R); Tyrosine (Y);    Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W);    Leucine (L); Lysine (K); Histidine (H); Cysteine (C), Glycine (G);    or is deleted;-   y & y′=is independently selected from Arginine (R); Tyrosine (Y);    Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W);    Leucine (L); Lysine (K); Cysteine (C); Histidine (H); or is deleted;-   z & z′=is independently selecled from Arginine (R); Tyrosine (Y);    Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W);    Leneine (L); Lysine (K); Cysteine (C); Histidine (H); or is deleted.

The polypeptide of formula I may comprise at least one additional aminoacid, which may be independendy selected from Arginine (R); Tyrosine(Y); Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W);Leucine (L); Lysine (K); Histidine (H). Preferably, the additional aminoacid is added before the amino acid at position ‘a’ in the peptide offormula I, i.e. to the N-terminal.

Hence, it will be appreciated that the polypeptide according to theinvention may comprise an 18-mer of {abcRKRxyx} and {a′b′c′RKRx′y′z′},in which abc, a′b′c′, xyz and x′y′z′ are defined as above, or atruncation thereof. It will be appreciated that, for example, a may bedifferent to a′, and b may be different to b′, and c may be different toc′, and so on.

The polypeptide according to the first aspect may preferably be ahomodimer of formula II:

{abcRKRxyz}+{abcRKRxyz}

wherein a, b, c, x, y annd z are as defined for formula I.

As with the polypeptide of formula I, the polypeptide of formula II maycomprise at least one additional amino acid, which may be independendyselected from Arginine (R); Tyrosine (Y); Methionine (M); Isoleucine(I); Phenylalanine (F); Tryptophan W; Leucine (L); Lysine (K); Histidine(H). Preferably, the additional amino acid is added before the aminoacid at position ‘a’ in the peptide of formula II i.e. to theN-terminal.

Hence, it will be appreciated that the polypeptide according to theinvention comprises an 18-mer of {abcRKRxyz} and {abcRKRxyz}, in which,abc and xyz are defined as above, or a truncation thereof.

The polypeptide defined by formula II preferably comprises the followingamino acids:

-   a=is independently selected from Arginine (R); Tyrosine (Y);    Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W);    Cysteine (C); or is deleted;-   b=is independently selected from Arginine (E); Tyrosine (Y);    Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W);    Cysteine (C); or is deleted;-   c=is independently selected from. Phenylalanine (F); or Tryptophan    (W); Cysteine (C); or is deleted;-   x=is independently selected from Phenylalanine (F); Tryptophan (W);    Cysteine (C); or is deleted;-   y=is independently selected from Phenylalanine (F); Tryptophan (W);    Cysteine (C); or is deleted;-   z=is independently selected from Arginine (R); Tyrosine (Y);    Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W);    Cysteine (C):or is deleted.

These preferred polypeptides may enrnprise at least one additional aminoacid, which may be either Phenylalanine (F) or Tryptophan (W) or Leucine(L). Preferably, the additional amino acid is added before the aminoacid at position ‘a’ in the polypeptide of formula II i.e. to theN-Terminal.

The inventors have also appreciated that polypeptides may be employedaccording to the invention that comprise more than just a tandem dimer(2×) repeat of a peptide derived from a Heparan Sulphate Proteoglycan(HSPG) receptor binding region of apolipoprotein B or apolipoprotein E,or a truncation thereof. For example, polypeptides comprising a trimer(3×) repeat, or tetramer (4×) repeat, or an even, greater number ofrepeats of a peptide derived, from a Heparan Sulphate Proteoglycan(HSPG) receptor binding region of spoiipoprotein B or apolipoprotein Emay be employed as useful antifungal and/or autiprotist agents.

Accordingly, it is preferred mat the polypeptide may have formula III:

{abcRKRxyz}^(n)

wherein a, b, c, x, y, and z are as defined above with reference toformula I or II, and wherein n is equal to 2, 3, 4 or 5, or more.

Other preferred polypeptides may comprise repeats of the 18-mer peptide(or truncation thereof) defined by formula I (e.g. repeats of ahetetodhner of the 9-mers comprising the peptide of formula I).

It is most preferred that the polypeptide according to the first aspectmay comprise a repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 5), or derivatives andtruncations thereof. Such peptides represent an important embodiment ofthe invention. Hence, in a second aspect, there is provided use of apolypeptide, derivative or analogue thereof comprising a repeat of thepeptide apoE₁₄₁₋₁₄₉ (SEQ ID NO. 5) or a truncation thereof; or a repeatof a variant of the peptide apoE₁₄₁₋₁₄₉ in which at least one Leucine(L) residue is replaced by Tryptophan (W), Arginine (R), Lysine (K),Tyrosine (Y) Cysteine (C) or Phenylalanine (F), for the manufacture of amedicament for the treatment of a fungal and/or protist infection, orcontamination.

By “a repeat of the peptide apoE₁₄₁₋₁₄₉”, we mean a polypeptidecomprising a repeat of the peptide sequence: LRKLRKRLL (SEQ ID No 5),i.e., a 9-mer. The polypeptide preferably comprises the amino acidsequence: LRKLRKRLLLRKLRKRLL (SEQ ID NO. 8), i.e. an 18-mer, which is atandem repeat diner of SEQ ID No.5. SEQ ID No. 8 is also referred toherein as GIN 1 or GIN1p (wherein p signifies N terminal protection(e.g. by an acetyl group), and C terminal protection (e.g. by an amidegroup)). GIN 1p is also referred to herein as MU 10.

By “a truncation thereof”, we mean that the repeat (e.g. the 18-mer ofSEQ ID No.8) is reduced in size by removal of amino acids. The reductionof amino acids may be by removal of residues from the C- and/orN-terminal, or may be by deletion of one or mom amino acids from withinthe core of the polypeptide (e.g. amino acids 2-17 of SEQ ID No.8).

The inventor has identified that Tryptophan (W), Arginlne (R), Lysine(K), Tyrosine (Y), Cysteine (C) or Phenylalanine (F) may be substitutedfor Leucine in apoE₁₄₁₋₁₄₉ tandem repeats, and that such polypeptideshave surprising antifungal/antiprotist activity.

It is more preferred that polypeptides according to the second aspect ofthe invention comprise a polypeptide, derivative or analogue thereofcomprising a dimer repeat of apoE₁₄₁₋₁₄₉ or a truncation threreof,characterised in that at least one Leucine (L) residue of the dimer ofSEQ ID No.8 is replaced by a Tryptophan (W), or a Phenylalanine (F)residue.

It is most preferred that at least one Leucine (L) residue of the dimerof SEQ ID No.8 is replaced by a Tryptophan (W) residue.

As discussed in more detail below, SEQ ID No.8 may be manipulated with anumber of different substitutions and deletions to make polypeptideswith antifungal, and/or antiprolisi activity. It is preferred that thepolypeptide according to the second aspect of the invention has at leasttwo substitutions independently selected from: Tryptophan (W), Arginine(R), Lysine (K), Tyrosine (Y), Cysteine (C) or Phenylalanine (F)substitutions, and more preferably three or more substitutions. It ispreferred that these multiple substitutions are with Tryptophan (W),Arginine (R), Lysine (K), Tyrosine (Y), or Phenylalanine (F) and mostpreferred that there are multiple substitutions with Tryptophan (W).

In addition, to one or more L substitutions with K, R, Y, F, C or W, itis preferred that at least one further amino acid (preferably at leastone further Leucine residue) is replaced with Arginine (R), Tyrosine(Y), Methionine (M), Isoleucine (I), Phenylalanine (F), or Tryptophan(W). It is particularly preferred that such a further substitution is For W.

The inventor has also appreciated that polypeptides may be employedaccording to the invention that comprise more than just a dimer tandemrepeat of ApoE₁₄₁₋₁₄₉ or a truncation or variant thereof. For instance,a trimer or tetramer or greater number of repeats may be employed asantifungal and/or antiprotistagents.

The polypeptides according to the second aspect may be synthesised suchthat further amino acids are added thereto. For instance, one, two,three or more amino acids may be added to the C or N terminals of apeptide of SEQ ID No.8 or a derivative of such a peptide as definedabove. Alternatively, the polypeptide may comprise a tandem repeat of apeptide that is larger than the nine amino acids of SEQ ID No.5. Suchpeptides may have amine acids added to the N terminal, C terminal and/orbetween the 9^(th) and 10^(th) amino acids of SEQ ID No. 8. It is mostpreferred that the amino acid is added to the C terminal and alsobetween the 9^(th) and 10^(th) amino acids of SEQ ID No.8. It will beappreciated that such peptides may then be modified as described abovefor polypeptides derived from SEQ ID No.8.

The substituted polypeptide may comprise 18 amino acids (or derivativesthereof) and thereby correspond to the full length of a dimer tandemrepeat of apoE₁₄₁₋₁₄₉. However, the inventors have surprisingly foundthat some selected truncated polypeptides based on SEQ ID No.8 also haveefficacy as antifungal and/or antiprotist agents. Accordingly, preferredpolypeptides or derivatives thereof may have less than 18 amino acids.For instance, some polypeptides according to the second aspect of theinvention may be 17, 16, 15, 14, 13, 12, 11, 10 or less amino acids inlength.

It will be appreciated that modified forms of W, Y, R, K, C or F may besubstituted into the tandem repeat of apoE₁₄₁₋₁₄₉ with a number of aminoacid variants that may be known to those skilled in the art. Suchpolypeptides will still have antifungal and/or antiprotist activityprovided that the modification does not significantly alter its chemicalcharacteristics. For instance, hydrogens on the side chain amines of Ror K may be replaced with methylene groups (—NH₂→—NH(Me) or —N(Me)₂).

Other preferred polypeptides according to the second aspect of theinvention (comprising tandem repeats of peptides derived fromapoE₁₄₁₋₁₄₉) may comprise one of the following amino acid sequences:

-   (a) WRKWRKRWWWRKWRKRWW (SEQ ID No. 9). This polypeptide corresponds    to a full length tandem dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 8)    with all Leucines substituted for Tryptophan residues. This    polypeptide is designated GIN 7 or MU 4 when referred to herein.-   (b) WRKWRKRWRKWRKR (SEQ ID No. 10). This polypeptide corresponds to    the full length tandem dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 8)    with all Leucines substituted for Tryptophan residues and truncated    by the excision of amino acids 9, 10, 17 and 18, i.e. is a 14-mer.    This polypeptide is designated GIN 32 when referred to herein.-   (c) WRKWRKRWWLRKLRKRLL (SEQ ID No. 11). This polypeptide corresponds    to the full length tandem, dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID    NO. 8) with a subset of Leucines substituted for Tryptophan    residues, i.e. is an 18-mer. This polypeptide is designated GIN 34    when referred to herein.-   (d) YRKYRKRYYYRKYRKRYY (SEQ ID No. 12). This polypeptide corresponds    to the full length tandem dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 8)    with all Leucines substituted for tyrosine residues, i.e. is an    18-mer. This polypeptide is designated GIN 41 or MU6 when referred    to herein.-   (e) LRKLRKRLRKLRKR (SEQ ID No. 13). This polypeptide corresponds to    the full length tandem dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 8)    truncated by the excision of amino acids 9, 10, 17 and 18, i.e. is    an 14-mer. This polypeptide is designated GIN 8 when referred to    herein.-   (f) LRKRLLLRKLRKRLL (SEQ ID No.14). This polypeptide corresponds to    the full length tandem dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 8)    truncated by the excision of amino acids 1, 2 and 3, i.e. is a    15-mer. This polypeptide is designated GIN 2 when referred to    herein.-   (g) FRKFRKRFFFRKFRKRFF (SEQ ID No.15). This polypeptide is    designated MU 7 when referred to herein.-   (h) WRKWRKRWWRKWRKRWW (SEQ ID NO.16). This polypeptide corresponds    to SEQ ID No. 9 with the W residue at position 9 deleted. This    polypeptide is designated MU 58 when referred to herein.-   (i) WRKWRKRWRKWRKRW (SEQ ID NO.17). This polypeptide corresponds to    SEQ ID No. 9 with the W residues at position 9, 10 and 18 deleted.    This polypeptide is designated MU 59 when referred to herein.-   (j) WRKWRKRWWFRKWRKRWW (SEQ ID NO.18). This polypeptide corresponds    to SEQ ID No. 9 with the W residue at position 10 substituted with    an F. This polypeptide is designated MU 60 when referred to herein.-   (k) WRKWRKRFFWRKWRKRFF (SEQ ID NO. 19). This polypeptide corresponds    to SEQ ID No. 9 with the W residues at positions 9, 10, 17 and 18    substituted with F residues. This polypeptide is designated MU 61    when referred to herein.-   (l) WRKRWWRWRKRWWR (SEQ ID NO.20). This polypeptide is designated MU    81 when referred to herein.-   (m) LRKLRKRLLRLRKLRKRLLR (SEQ ID ND.21). This polypeptide is    designated MU 82 when referred to herein.-   (n) WRKWRKRWWRWRKWRKRWWR (SEQ ID NO.22). This polypeptide is    designated MU 83 when referred to herein.-   (o) LRKLRKRLLWRKWRKRWW (SEQ ID NO.23). This polypeptide corresponds    to SEQ ID No. 8 with the L residues at positions 10, 13, 17 and 18    substituted with W residues. This polypeptide is designated MU 111    when referred to herein.-   (p) LRKLRKRLLLRKLRKRWW (SEQ ID NO.24). This polypeptide corresponds    to SEQ ID No. 8 with the L residues at positions 17 and 18    substituted with W residues. This polypeptide is designated MU 112    when referred to herein.-   (q) LRKLRKRLLWRKWRKRLL (SEQ ID NO.25). This polypeptide corresponds    to SEQ ID No. 8 with the L residues at positions 10 and 13    substituted with W residues. This polypeptide is designated MU 113    when referred to herein.-   (r) WRKWRKRLLLRKLRKRLL (SEQ ID NO.26). This polypeptide corresponds    to SEQ ID No. 8 with the L residues at positions 1 and 4 substituted    with W residues. This polypeptide is designated MU 114 when referred    to herein.-   (s) WRKLRKRLLLRKLRKRLL (SEQ ID NO.27) This polypeptide corresponds    to SEQ ID No. 8 with the L residue at position 1 substituted with W    residues. This polypeptide is designated MU 115 when referred to    herein.-   (t) WRKWRKFFFRKWRKRWW (SEQ ID NO.28). This polypeptide corresponds    to SEQ ID No. 9 with the W residues at positions 8, 9 and 10    substituted with F residues and the R residue at position 7 deleted.    This polypeptide is designated MU 116 when referred to herein.-   (u) WRKWRKRWWFRKFRKRFF (SEQ ID NO.29). This polypeptide corresponds    to SEQ ID No. 9 with the W residues at positions 10, 13, 17 and 18    substituted with F residues. This polypeptide is designated MU 117    when referred to herein.-   (v) CRKCRKRCCCRKCRKRCC (SEQ ID No. 30). This polypeptide corresponds    to the full length tandem dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 8)    with all Leucines substituted for cysteine residues, i.e. is an    18-mer. This polypeptide is designated MU 12 when referred to    herein.-   (w) RRKRRKRRRRRKRRKRRR (SEQ ID No. 31). This polypeptide corresponds    to the full length tandem dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 8)    with all Leucines substituted for arginine residues, i.e. is an    18-mer. This polypeptide is designated MU 16 when referred to    herein.-   (x) MRKMRKRMMMRKMRKRMM (SEQ ID No. 32). This polypeptide corresponds    to the full length tandem dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 8)    with all Leucines substituted for methionine residues, i.e. is an    18-mer. This polypeptide is designated MU 5 when referred to herein.-   (y) IRKIRKRIIIRKIRKRII (SEQ ID No. 33). This polypeptide corresponds    to the full length tandem dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 8)    with all Leucines substituted for isoleueine residues, i.e. is an    18-mer. This polypeptide is designated MU 8 when referred to herein.-   (z) HRKHRKRHHHRKHRKRHH (SEQ ID No. 34). This polypeptide corresponds    to the full length tandem dimer repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO. 8)    with all Leucines substituted for histidine residues, i.e. is an    18-mer. This polypeptide is designated MU 19 when referred to    herein.

In most preferred embodiments, polypeptides according to the secondaspect of the invention (comprising tandem repeats of peptides derivedfrom apoE₁₄₁₋₁₄₉) comprise one of the following amino acid sequences:

-   -   (1) WRKWRKRWWWRKWRKRWW (SEQ ID No. 9). This polypeptide        corresponds to a full length tandem dimer repeat of apoE₁₄₁₋₁₄₉        (SEQ ID NO. 8) with all Leucines substituted for Tryptophan        residues. This polypeptide is designated GIN 7 or MU 4 when        referred to herein;    -   (ii) FRKFRKRFFFRKFRKRFF (SEQ ID No.15). This polypeptide is        designated MU 7 when referred to herein;    -   (iii) LRKLRKRLLLRKLRKRLL (SEQ ID NO. 8), i.e. an 18-mer, which        is a tandem repeat dimer of SEQ ID No.4, SEQ ID No. 8 is also        referred to herein as GIN 1 or GIN1p (wherein p signifies N        terminal protection (e.g. by an acetyl group), and C terminal        protection (e.g. by an amide group). GIN 1p is also referred to        herein as MU 10; and    -   (iv) WRKWRKRLLLRKLRKRLL (SEQ ID NO.26). This polypeptide        corresponds to SEQ ID No. 4 with the L residues at positions 1        and 4 substituted with W residues. This polypeptide is        designated MU 114 when referred to herein.

Furthermore QSTEELRVRLASHLRKLRKRLL (SEQ ID No. 35), which contains onlyone RKR motif has been found to be a useful antifungal agent. Thispolypeptide is designated GIN 11 when referred to herein.

According to another embodiment of the first aspect of the inventionpreferred polypeptides comprise repeats of peptides derived from an HSPGreceptor binding region of apolipoprotein B, or a variant or truncationthereof. Hence, in a third aspect there is provided use of apolypeptide, or a derivative or analogue thereof, comprising repeats ofa peptide derived from an HSPG receptor binding region of apolipoproteinB, for the manufacture of a medicament for the treatment of a fungaland/or protist infection, or contamination.

Preferably, the polypeptide, derivative or analogue thereof comprises arepeat which is derived from an apolipoprotein B LDL receptor bindingdomain cluster B. Preferably, the polypeptide, derivative or analoguethereof comprises a repeat of the peptide apoB₃₃₅₉₋₃₃₆₇ (SEQ ID No. 6)or a truncation or variant thereof.

The polypeptide according to the third aspect of the invention may be atandem dimer repeat of apoB₃₃₅₉₋₃₃₆₇ (SEQ ID No. 6) with the amino acidsequence: RLTRKRGLKRLTRKRGLK, i.e. an 18-mer (SEQ ID No.36).

Peptides according to the third aspect of the invention may also betruncated as defined herein. The reduction of amino acids may be byremoval of residues from the C- and/or N-terminal, or may be by deletionof one or more amino acids from within the core of the peptide (i.e.amino acids 2-17 of SEQ ID No.36),

It is preferred that polypeptides according to the third aspect compriseat least two RKR motifs, or more if the polypeptide is a trimer, ortetramer, and so on.

Preferred polypeptides according to the third aspect comprises thetandem dimer repeat of the peptide apoB₃₃₅₉₋₃₃₆₇ (i.e. the polypeptideof SEQ ID No. 36) or a truncation thereof, characterised in that atleast one amino acid residue, other than the RKR motifs, has beenreplaced by a Glycine (G), Threonine (T), Histidine (H), Tryptophan (W),Arginine (R) or Leucine (L) residue or derivatives thereof.

Suitably, one or more, more suitably, two or more, and even moresuitably, three or more amino acid residues may be replaced by a Glycine(G), Threonine (T), Histidine (B), Tryptophan (W), Arginine (R) orLeucine (L) residue or derivative thereof. Preferably, four or more,more preferably, five or more, and even more preferably, six or moreamino acid residues may be replaced by these ammo acids or derivativethereof. Preferably, the replaced or substituted residue is the first,second, third, seventh, eighth, ninth, tenth, eleventh, twelfth,sixteenth, seventeenth or eighteenth residue of SEQ ID No.36.

Preferably, the polypeptide according to the third aspect comprises thepolypeptide of SEQ ID No.36 or a truncation thereof, characterised inthat at least one amino acid residue has been replaced by a Tryptophan(W), Arginine (R) or Leucine (L) residue or derivative thereof.

Suitably, one or more, more suitably, two or more, and even moresuitably, three or more amino acid residues may be replaced by aTryptophan (W), Arginine (R) or Leucine (L) residue or derivativethereof. Preferably, four or more, more preferably, five or more, andeven more preferably, six or more amino acid residues may be replaced bya Tryptophan (W), Arginine (R) or Leucine (L) residue or derivativethereof. Preferably, the replaced or substituted residue is the first,second, third, seventh, eighth, and/or ninth residue of the repeatedamino acid sequence of apoB₃₃₅₉₋₃₃₆₇ or combinations thereof.

The polypeptide according to the invention may comprise 18 amino acids(or derivatives thereof) and thereby correspond to the full length ofSEQ ID No.36 with or without the substitutions discussed above. However,the inventors have surprisingly found that truncated polypeptides basedon SEQ ID No.36 also have efficacy as antifungal and/or antiprotistagents. Accordingly, preferred polypeptides or derivatives thereof mayhave less than 18 amino acids. For instance, some polypeptides accordingto the third aspect of the invention may be 17, 16, 15, 14, 13, 12, 11,10 or less amino acids in length. Deletions are preferably made atpositions 1, 2, 8, 9, 10, 11, 17 and/or 18 of SEQ ID No.36.

In a preferred embodiment, the polypeptide according to the third aspectmay preferably have formula IV:

{abcRKRxyz}+{a′b′c′RKRx′y′z′}

wherein

-   a & a′=is independently selected from a positively charged residue,    which may be selected from either Arginine (R) or Lysine (K) or    Histidine (H); Leucine (L); Tryptophan (W); or is deleted;-   b & b′=is independently selected from Leucine (L); Arginine (R);    Lysine (K); or is deleted;-   c & c′=is independently selected from Threonine (T); Tryptophan.    (W); or a positively charged residue, which may be selected from    Arginine (R) or Lysine (K) or Histidine (H);-   x &x′=is independently selected from Glycine (G); Tryptophan (W);    Leucine (L); or a positively charged residue, which may be selected    from Arginine (R) or Lysine (K) or Histidine (H);-   y & y′=is independently selected from Leucine (L); a positively    charged residue, which may be selected, from Arginine (R) or    Lysine (K) or Histidine (H); or is deleted;-   z &z′=is independently selected from a positively charged, residue,    which, may be selected from Arginine (R) or Lysine (K) or Histidine    (H); or Leucine; or is deleted.

The polypeptide according to the third aspect may also preferably haveformula V:

{abcRKRxyz}+{abcRKRxyz}

wherein

-   a=is independently selected from a positively charged residue, which    may be selected from either Arginine (R) or Lysine (K) or Histidine    (H); Leucine (L); Tryptophan (W); or is deleted;-   b=is independently selected from Leucine (L); Arginine (R); Lysine    (K); or is deleted;-   c=is independently selected from Threonine (T); Tryptophan (W); or a    positively charged residue, which may be selected from Arginine (R)    or Lysine (K) or Histidine (H);-   x=is independently selected from Glycine (G); Tryptophan (W);    Leucine (L); or a positively charged residue, which may be selected    from Arginine (R) or Lysine (K) or Histidine (H);-   y=is independently selected from Leucine (L); a positively charged    residue, which may be selected from Arginine (R) or Lysine (K) or    Histidine (H); or is deleted;-   z=is independently selected from a positively charged residue, which    may be selected from Arginine (R) or Lysine (K) or Histidine (H); or    Leucine (L); or is deleted.

The polypeptide of formula V may more preferably comprise the followingamino acids:

-   a=is independently selected from Trypotphan (W); Arginine (R);    Leucine (L); or is deleted;-   b=is independently selected from Leucine (L); Arginine (R) or Lysine    (K); or is deleted;-   c=is independently selected from Tryptophan (W); Trheonine (T);    Lysine (K);-   x=is independently selected from Tryptophan (W); Glycine (G);    Leucine (L); Arginine (R);-   y=is independently selected from Leucine (L); a positively charged    residue, which may be selected from Arginine (R) or Lysine (K) or    Histidine (H); or is truncated here;-   z=is independently selected from a positively charged residue, which    may be selected from Arginine (R) or Lysine (K) or Histidine (H); or    Leucine (L); or is truncated here.

The inventors have also appreciated that polypeptides may be employedaccording to the invention that comprise more than just a tandem dimerrepeat of apoB₃₃₅₉₋₃₃₆₇ (SEQ ID No.36) or a variant or truncationthereof. For example, polypeptides comprising a trimer, or tetramer, oreven greater number of repeats of SEQ ID No.6 may be employed as usefulantifungal/protist agents.

Accordingly, it is preferred that the polypeptide may preferably haveformula VI:

{abcRKRxyz}^(n)

wherein a, b, c x, y, and z are as defined above with reference toformula IV or V, and wherein n is equal to 2, 3, 4 or 5, or more. Itwill bo appreciated that monomer peptides {abcRKRxyz} may be identicalor may vary as defined-above.

Other preferred, polypeptides may comprise repeats of the 18mer (ortruncations thereof) defined by formula XV or V (e.g. repeats of aheterodimer of the 9mer peptides defined by formula IV).

Other preferred polypeptides according to the third aspect of theinvention may comprise one of the following amino acid sequences:

-   a) RTRKRGRRTRKRGR (SEQ ID No.37). This polypeptide is designated GIN    36 when referred to herein;-   b) LRKRKRLLRKRKRL (SEQ ID No.38). This polypeptide is designated GIN    37 when referred to herein;-   c) LRKRKRLRKLRKRKRLRK (SEQ ID No.39). This polypeptide is designated    GIN 38 when referred to herein;-   d) WRWRKRWRKWRWRKRWRK (SEQ ID No.40). This polypeptide is designated    GIN 33 when referred to herein;-   e) LLRKRLKRLLLRKRLKRL (SEQ ID NO.41). This polypeptide is designated    MU 24 when referred to herein;-   f) RRWRKRWRKWRWRKRWRK (SEQ ID NO.42). This polypeptide is designated    MU 28 when referred to herein;-   g) KRWRKRWRKWRWRKRWRK (SEQ ID NO.43). This polypeptide is designated    MU 29 when referred to herein;-   h) LRWRKRWRKWRWRKRWRK (SEQ ID NO.44). This polypeptide is designated    MU 30 when referred to herein;-   i) HRWRKRWRKWRWRKRWRK (SEQ ID NO.45). This polypeptide is designated    MU 31 when referred to herein;-   j) RWRKRWRKWRWRKRWRK (SEQ ID NO.46). This polypeptide is designated    MU 32 when referred to herein;-   k) RRWRKRWRKRRWRKRWRK (SEQ ID NO.47). This polypeptide is designated    MU 33 when referred to herein;-   l) LRWRKRWRKLRWRKRWRK (SEQ ID. NO.48). This polypeptide is    designated MU 35 when referred to herein;-   m) HRWRKRWRKHRWRKRWRK (SEQ ID NO.49). This polypeptide is designated    MU 36 when referred to herein;-   n) RWRKRWRKRWRKRWRK (SEQ ID NO.50). This polypeptide is designated    MU 37 when referred to herein;-   o) RWRKRGRKRWRKRGRK (SEQ ID NO.51). This polypeptide is designated    MU 69 when referred to herein;-   p) RWRKRWRKRWRKRWRK (SEQ ID NO.52). This polypeptide is designated    MU 71 when referred to herein;-   q) RKRGWKWRKRGWKW (SEQ ID NO.53). This polypeptide is designated MU    73 when referred to herein;-   (r) RLTRKRGRLTRKRG (SEQ ID NO.54). This polypeptide is designated MU    74 when referred to herein; and-   (s) WRWRKRWRKWRWRKRWRK (SEQ ID NO.55). This polypeptide is    designated MU 27 when referred to herein;

Derivatives of polypeptides according to the invention may be used totreat fengal and/or protist infections. Such derivatives may increase ordecrease the polypeptide's half-life in vivo. Examples of derivativescapable of increasing the half-life of polypeptides according to theinvention include peptoid derivatives of the polypeptides. D-amino acidderivatives of the polypeptides, and peptide-peptoid hybrids.

Polypeptides according to the invention may be subject to degradation bya number of means (such as protease activity in biological systems).Such degradation may limit the bioavailability of the polypeptides andhence the ability of the polypeptides to achieve their biologicalfunction. There are wide ranges of well-established techniques by whichderivatives that have enhanced stability in biological contexts can bedesigned and produced. Such polypeptide derivatives may have improvedbioavailability as a result of increased resistance to protease-mediateddegradation. Preferably, a derivative or analogue suitable for useaccording to the invention is more protease-resistant than the peptidefrom which it is derived.

Preferably, the polypeptide may be made more protease-resistant byprotecting the N and/or C terminal. For example, the N terminal may beprotected by an acetyl group, or by an alkyl or aryl group, or analkyl-CO— or aryl-CO— group, each of which may be optionallysubstituted. The C terminal may be protected by an amide group or by asubstituted amide group.

Protease-resistance of a polypeptide derivative and the polypeptide fromwhich it is derived may be evaluated by means of well-hnown proteindegradation assays. The relative values of protease resistance for thepolypeptide derivative and polypeptide may then be compared.

Peptoid derivatives of the polypeptides of the invention may be readilydesigned from knowledge of the structure of the polypeptide according tothe first, second or third aspect of the invention. Commerciallyavailable software may be used to develop peptoid derivatives accordingto well-established protocols.

Retropeptolds, (in which all amino acids are replaced by peptoidresidues in reversed order) are also able to mimic antibacterialpolypeptides derived from apolipoproieins. A retropeptoid is expected tobind in the opposite direction in the ligand-binding groove, as comparedto a peptide or peptoid-peptide hybrid containing one peptoid residue.As a result, the side chains of the peptoid residues are able to pointin the same direction as the side chains in the original peptide.

A further embodiment of a modified form of polypeptide according to theinvention comprises D-amino acid forms of the polypeptide. Thepreparation of peptides using D-amino acids rather than L-amino acidsgreatly decreases any unwanted breakdown of such an agent by normalmetabolic processes, decreasing the amounts of agent which need to beadministered, along with the frequency of its administration.

Other modifications in polypeptide sequences are also envisaged andwithin the scope of the claimed invention, i.e. those which occur duringor after translation, e.g. by acetylation, amidation, carboxylation,phosphorylation, proteolytic cleavage or linkage to a ligand.

The inventor believes that polypeptides, derivatives or analoguesaccording to the invention may be used in the prevention or treatment ofany fungal or protist infection. According to one preferred embodimentof the invention medically important species (e.g. animals and man) maybe treated according to the first second or third aspects of theinvention to prevent or treat an infection cause by a fungus. By theterm “fungus”, we mean any of the numerous eukaryotic organisms of thekingdom Fungi. These tend to lack chlorophyll, and may range in formfrom single cellular to multicellular, and may be branched filamentoushyphae that often produce fruiting bodies. Hence, the fungus may befilamentous.

Examples of fungal species, which cause medically important fungalinfections (for example in man or in veterinary situations), and whichmay be treated by the peptides according to the invention may beindependently selected from a group consisting of:—Chytridiomycota;Zygomycota; Ascomycota; Basidiomycota; Lichens; Deuteromycota;Mitosporidia; and Straminipila.

Preferred Chytridiomycota against which the peptide according to theinvention may have activity may be indepondendy selected from a groupconsisting of:—Neocallimasticales; Blastocladiales; Chytriddiales;Spizellomycetales; and Monoblepharidales.

Preferred Zygomycota against which the peptides according to theinvention may have activity may be independently selectedfrom:—Mucorales; or Entomophthorales.

Preferred Basidiomycota against which the peptides are active may beindependently selected from a group consisting of:—Sporidales, andHymenomycetes, Preferred sporidiales may include Cryptococcusneoformans, and preferred Hymenomycetes may include Malassezia spp.

Preferred Mucorales against which the peptides according to theinvention are active may be independently selected from a groupconsisting of:—Musoraceae; Absidia; Apophysomyces; Mucor; Rhizomucor;Syncephalastraceae; Mortierellaceae; Saksenaeaceae; Thamnidiaceae; andCunninghamellaceae.

Preferred Mucoraceae against which the peptides are active may includeRhizopus, for example, Rhizopus arrhizus. Preferred Absidia includeAbsidia corymbifera. Preferred Apophysomyces include Apophysomyceselegans. Preferred Syncephalastraceae include Syncephalastrum racemosum.Preferred Saksenaeaceae inlcude Saksenaea vasiformis. PreferredThamnidiaceae include Cokeromyces recurvatus. PreferredCunninghamellaceae include Cunninghamella bertholletiae.

Preferred Entomophthorales against which the peptides according to theinvention are active may be independently selected from a groupconsisting of:—Basidiobcdaceae; Entomophthoraceae; Completoriaceae;Ancylistaceae; Meristacracceae; and Neozygitaceae. PreferredBasidiobolaceae include Basidiobolus ranarum and Lacazia loboi.Preferred Ancylistaceae include Conidiobolus coronatus and Conidiobolusincongruus.

Preferred Asconycota against which the peptides are active may beindependently selected from a group consisting of: Ascomycetes andEndomyetes.

Preferred Asconmycetes against which the peptides according to theinvention are active may be independently selected from a groupconsisting of:—Onygenales; Histoplasma spp.; Onygenaceae;Laboulbeniomycetes, Protoascomycetes; Euascomycetes; Chaetothyriales;Ascomycotina; Paracoccidioides; Cladosporium; Endomycetes;Saccharomycetales; Dipodascaceae; and Saccharomycetaceae.

Preferred Onygenales include Arthodermataceae, for example,Epidermophyton spp.; Microsporum spp. and Trichophyton spp. PreferredHistoplasma spp. include Histoplasma capsulatum. Preferred Euascomycetesmay be independently selected from a group consisting of Bipolaris spp,Blastomyces dermatitidis, Coccidioides immitis, Coccidioides posadasii,Curvularia spp., Fonsecaea, Leptosphaeria spp., Madurella, Neotestudinaspp., Phialophora, Piedraia spp., Pseudallescheriam, Pyrmochaeta,Scedosporium spp., Scopulariopsis spp. and Sporothrix schenckii.Preferred Chaetothyriales include Exophiala spp. and Wangiella spp.Preferred Ascomycotina include Acremonium spp. PreferredParacoccidioides include Paracoccidioides brasiliensis. PreferredEndomycetes include Saccharomycetales, including Dipodascaceae andSaccharomycetaceae. Preferred Dipodascaceae include Dipodascus andarthroconidia.

Most preferred Saccharomycetaceae against which the peptides accordingto the invention are active may be independently selected from a groupconsisting of:—Candida; Eurotiales; and Hypocreales.

Examples of preferred Candida spp against which the peptides accordingto the invention may be active may be independently selected from agroup consisting of:—Candida tropicalis; Candida glabrata; Candidaparapsilosis; Candida krusei; Candida lusitaniae; and most preferably,Candida albicans. A most preferred Candida albicans is Candida albicans6862.

Preferred Eurotiales include Aspergillus spp. Preferred Aspergillusagainst which the peptides according to the invention may be active maybe independently selected from a group consisting of:—Aspergillusflavus, Aspergillus fumigatus; Aspergillus glaucus; Aspergillusnidulans; Aspergillus niger; and Aspergillus terreus, A most A.fumigatus is AF293.

Preferred Hypocreales include Fusarium spp. Preferred Fusarium againstwhich the peptides according to the invention may be active may beindependently selected from a group consisting of:—Fusarium solani;Fusarium oxysporum; and Fusarium chlamydosporium. Most preferredFusarium spp include either Fusarium spp 5889 or Fusarium spp 6507.

Polypeptides, derivatives or analogues according to the invention may beused in the treatment against any protlsi or protist infection, orcontamination therewith. By the term “protist”, we mean any of thenumerous generally unicellular eukaryotic organisms of the kingdomProtista. However, it will be appreciated that some protists aremulticellular. The prolist may be a protozoan. Some forms of Protistaare responsible for causing disease, especially in humans.

For example, preferred protists (or Protocista) against which thepeptides in accordance with the present invention are elective may beindependently selected from a group consisting of: Chlorophyta (GreenAlgae); Phaeophyta (Brown Algae); Pyrrophyta (Dinoflagellates);Chrysophyta (Diatoms); Rhodophyta (Red Algae); Charophyta (Stoneworts);and Euglenophyta (Euglena).

Further examples of preferred protists include organisms within thePhylum Apicomplexa, such as, organisms independently selected from agroup consisting of:—Coccidia; Hemogregarina spp.; Eimeria; Isospora;Sarcocystis cruzi; Toxoplasma spp.; Cryptosporidium spp.: and Cyclosporacayetanensis.

Further preferred examples include Haemosporoina. Most preferredHaemosporoina include Plasmodium spp. it will be appreciated thatPlasmodium spp is the protist responsible for carrying and transmittingmalaria, a disease, which causes millions of deaths annually. PreferredPlasmodium spp may be independently selected from a group consisting ofPlasmodium vivax; Plasmodium malariae; Plasmodium ovale; and mostpreferably, Plasmodium falciparum.

Preferred Isospora include Isospora belli. Preferred Taxoplasma spp.include Toxoplasma gondil. Preferred Cryptosporidium spp. includeCryptosporidium parvum.

Further preferred examples of protists against which the peptides inaccordance with the present invention are effective may includeorganisms within the Phylum myxozoa, for example, Myxobolus cerebralis.

Further preferred examples of protists against which the peptides inaccordance with the present invention are effective may includeorganisms within the Phylum Ciliophora. Preferred Ciliophora againstwhich the peptides in accordance with the present invention areeffective may be independently selected from a group consistingof:—Ichthyophthirius multifiliis; and Trichodina sp. and those withinthe Class Litostomatea, including Balantidium coli.

Further preferred examples of protists against which the peptides inaccordance with the present invention are effective may includeorganisms within the Phylum Sarcomastigophora. These organisms mayinclude: (i) those within the suhphylum Mastigophora (the flagellates);and (ii) those within the subphylum Sarcodina.

Preferred examples of organisms within the subphylum Mastigophora may beindependently selected from a group consisting of Chilomastix mesnili;Dientamoeba fragilis; Trichomonoas vaginalias; Giardia lamblia;Cryptobia salmositica; Leishmania spp; and Trypanosoma spp, for example,Trypanosoma cruzi.

Preferred examples of organisms within the subphylum Sarcodina may beindependently selected from a gronp consisting of: Entamoebahistolytica; and free-living amoeba, for example, Naegleris Fowleri,Balamuthia mandriliaris, and Acanthamoeba spp. Preferred Acanthamoebaspp against which the peptides in accordance with the present inventionare effective may include organisms may be independently selected from agroup consisting of:—A. astronyxis; A. comandoni; A. divionensis; A.griffini; A. hatchetti; A. healyl; A. jacobsi; A. lenticulata; A.culbertsoni; A. lugdunensis; A. mauitaiensis; A. palestinensis; A.pearcei; A. polyphaga; A. pustulosa; A. quina; A. rhysodes; A. royreba;A. terricola; A. triangularis; A. tubiashi; A. polyphaga; and A.castellanii.

The inventor conducted experiments (Example 1) to determine antifungalactivity against the test fungi Aspergillus fumigatus AF293, Aspergillusniger, Aspergillus terreus, Candida albicans 6862, Fusarmm spp 5889) andFusarium spp 6507. The antifungal activity of polypeptides in accordancewith the invention can be seen in Table 2. Hence, preferably, thepolypeptides according to the invention exhibit antifungal activityagainst at least one, preferably at least two, and more preferably, allof Aspergillus spp. Candida spp., and Fusarium spp. Preferably, thepolypeptides according to the invention exhibit antifungal activityagainst all of Aspergillus, Candida albicans 6862, Fusarium spp 5889,and Fusarium spp 6507.

The inventors have found that MU4, MU10, and MU114 are particularlyactive against Aspergillus spp, in particular, A. fumigatus. AF293,Aspergillus niger, and Aspergillus terreus. In addition, the inventorshave found that MU4, MU10, and MU114 are particularly effective againstCandida spp., and in particular, Candida albicans. Furthermore, theinventors found that MU4, MU10, and MU114 are particularly effectiveagainst Fusarium spp., and in particular, F. graminarium.

In addition to testing the activity of polypeptides disclosed herein tokill fungi, the inventor also conducted experiments (Example 2) todetermine antiprotist activity against the test protists, i.e.Acanthamoeba polyphaga (trophozoites). As can be seen in Table 3, allthree peptides in accordance with the invention (MU4, MU7, MU10, andMU114 under test) were reactive against Acanthamoeba, and in particular,Acanthamoeba polyphaga.

Further preferred, antifungal and anti-protist applications aredescribed in Examples 4 and 5 respectively.

Polypeptides according to the invention, may be used to treat fungaland/or protist infections as a monotherapy (i.e. use of the polypeptideas the only anti-microbial) or in combination with other compounds ortreatments used in antifungal or antiprotist therapy. For example, thepolypeptides may be combined with conventional antifungal agents suchas: Amorolfine, Butanafine, Niaftifine, Terbinafine, Flucytosine,Fluconazole, Butoconazole, Itraconazole, Ketoeonazole, Posaconazole,Ravuconazole, Voriconazole, Clotrimazole, Econazole, Miconazole,Oxiconazole, Sulconazole, Terconazole, Tioconazole, Nikkomycin Z,Caspofungin, Micafungin (FK463), Anidulafungin (LY303366), AmphotericinB (AmB), AmB lipid Complex, AmB Colloidal Dispersion, liposomal AmB, AmBOral Suspension, Liposomal Nystatin, Topical Nystatin, Pimaricin,Griseofulvin, Ciclopiroxolamine, Haloprogin, Tolnaftate, Undecylenate.

Alternatively the polypeptides may be combined with conventionalantiprotist agents such as: propamidine isethionate, broline, imidazoles(e.g. miconazole), topical aminoglycosides (e.g. neomycin), and topicalantiseptics (e.g. polyhexamethylene biguanide), chlohexidinepropamidine, Chloroquine, Fansidar (Pyrimethamine, Sulfadoxine)Amodiaqnine Quinine/Quinidine, Halofantrine, Mefloquine,Artemether/Artesunate, Malarone, Chloroquine, Proguanil, andDoxycycline.

Polypeptides according to the invention may be formulated incompositions having a number of different forms depending, inparticular, on the manner in which the polypeptide is to be used. Thus,for example, the composition may be in the form of a powder, tablet,capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray,micelle, transdermal patch, liposome or any other suitable form that maybe administered to a person or animal. It will be appreciated that thevehicle of the composition of the invention should be one which is welltolerated by the subject to whom it is given, and preferably enablesdelivery of the polypeptides or derivatives to a target tissue.

Compositions comprising polypeptides, agents, nucleic acids orderivatives according to the invention may be used in a number of ways.For instance, oral administration may be required in which case thecompound may be contained within a composition that may, for example, beingested orally in the form of a tablet, capsule or liquid.Alternatively, the composition may be administered systemically byinjection into the blood stream. Injections may be intravenous (bolus orinfusion) or subcutaneous (bolus or infusion). The compounds may beadministered by inhalation (e.g. intranasally).

Compositions comprising polypeptides according to the invention may beorally administered or systemically administered. Furthermore,compositions may be administered by aerosol, for example, using anatomiser, winch may be administered nasally, or by an inhaler via thelungs. Alternatively, the compositions may be topically applied, forexample, in the form of a cream or gel. Topical administration is usefulwhen a subject to be treated has a bacterial skin infection. Thecomposition may be applied intravaginally (for example, if required toprotect the subject from sexually transamitted diseases), or rectally.

The polypeptides and derivatives thereof may also be incorporated withina slow or delayed release device. Such devices may, for example, beinserted on or under the skin, and the compound may be released overweeks or even months. Such devices may be particularly advantageous whenlong-term treatment with a polypeptide or derivative according to theinvention is required and which would normally require frequentadministration (e.g. at least daily injection).

It will be appreciated that the amount of a polypeptide or derivativethat is required is determined by its biological activity andbioavailability which in turn depends on the mode of administration, thephysicochemical properties of the polypeptide, agent, nucleic acid orderivative employed and whether the polypeptide, agent, nucleic acid orderivative is being used as a monotherapy or in a combined therapy. Thefrequency of administration will also be influenced by theabove-mentioned factors and particularly the half-life of thepolypeptide, agent, nucleic acid or derivative within the subject beingtreated.

Optimal dosages to be administered may be determined by those skilled inthe art, and will vary with the particular polypeptide in use, thestrength of the preparation, the mode of administration, the type ofinfection being treated or prevented and the advancement of the diseasecondition. Additional factors depending on the particular subject beingtreated will result in a need to adjust dosages, including subject age,weight, gender, diet, and time of administration.

A skilled person will appreciate that a knowledge of the IC₅₀ for thepolypeptides will allow him or her to calculate the concentration ofpolypeptide in a particular formulation and also the amount of apolypeptide that should be administered to a subject in need oftreatment. The inventor has found that polypeptides, and derivativesthereof, according to the invention preferably have an efficacy forinhibiting fungal growth such that their IC₅₀ value is about 75 μM orless, more preferably, about 60 μM or less, even more preferably, about50 μM or less, and still, more preferably, about 40 μM or less. However,it is preferred that the IC₅₀ value is about 30 μM or less, morepreferably, about 20 μM or less, and most preferred about 10 μM or less.In fact, in the case of at least some of the peptides according to theinvention, the inventor was most surprised to establish that IC50 valuesof about 5 μM or less, and even of about 2.5 μM or less were obtainable(e.g. MU4). The skilled technician will appreciate how IC₅₀ values maybe calculated for fungi.

Polyperitides, and derivatives thereof, according to the inventionpreferably have an efficacy for inhibiting protist growth such thattheir IC₅₀ value is about 250 μM or less. It is more preferred that theIC₅₀ value for inhibiting growth of protists is about 100 μM of less,more preferably, about 50 μM or less, and most preferably, about 40 μMor less. As above, the skilled technician will appreciate how IC₅₀values may be calculated for protists.

Known procedures, such as those conventionally employed by thepharmaceutical industry (e.g. in vivo experimentation, clinical trials,etc.), may be used to establish specific formulations of polypeptides orderivatives according to the invention and precise therapeutic regimes(such as daily doses and the frequency of administration).

Generally, a daily dose of between 0.01 μg/kg of body weight and 0.5g/kg of body weight of polypeptides or derivatives according to theinvention may be used for the prevention and/or treatment of a viralinfection, depending upon which specific polypeptide, agent, nucleicacid or derivative is used. More preferably, the daily dose is between0.01 mg/kg of body weight and 200 mg/kg of body weight, and mostprefefebly, between approximately 1 mg/kg and 100 mg/kg.

Daily doses may be given as a single administration (e.g. a single dailyinjection). Alternatively, the polypeptide, or derivative thereof usedmay require administration twice or more tiroes during a day. As anexample, polypeptides according to the invention may be administered astwo (or more depending upon the severity of the condition) daily dosesof between 25 mg and 7000 mg (i.e. assuming a body weight of 70 kg). Apatient receiving treatment may take a first dose upon waking and then asecond dose in the evening (if on a two dose regime) or at 3 or 4 hourlyintervals thereafter. Alternatively, a slow release device may be usedto provide optimal doses to a patient without the need to administerrepeated doses.

This invention provides a pharmaceutical composition comprising atherapeutically effective amount of a polypeptide or derivativeaccording to the invention and optionally a pharmaceutically acceptablevehicle. In one embodiment, the amount of the polypeptide or derivativethereof is an amount from about 0.01 mg to about 800 mg. In anotherembodiment, the amount of the polypeptide, agent, nucleic acid orderivative is an amount from about 0.01 mg to about 500 mg. In anotherembodiment, the amount of the polypeptide or derivative is an amountfrom about 0.01 mg to about 250 mg. In another embodiment, the amount ofthe polypeptide or derivative is an amount from about 0.1. mg to about60 mg. In another embodiment, the amount of the polypeptide orderivative is an amount from about 0.1 mg to about 20 mg.

This invention provides a process tor making a pharmaceuticalcomposition comprising combining a therapeutically effective amount of apolypeptide or derivative thereof according to the invention and apharmaceutically acceptable vehicle. A “therapeutically effectiveamount” is any amount of a polypeptide or derivative according to theinvention which, when administered to a subject provides preventionand/or treatment of a fungal and/or protist infection. A “subject” maybe a vertebrate, mammal, domestic animal or human being.

A “pharmaceutically acceptable vehicle” as referred to herein is anyphysiological vehicle known to those of ordinary skill in the art usefulin formulating pharmaceutical compositions.

In a preferred embodiment, the pharmceutical vehicle is a liquid and thepharmaceutical composition is in the form of a solution. In anotherembodiment, the pharmaceutically acceptable vehicle is a solid and thecomposition is in the form of a powder or tablet. In a furtherembodiment, the pharmaceutical vehicle is a gel and the composition isin the form of a cream or the like.

A solid vehicle can include one or more substances, which may also actas flavouring agents, lubricants, solubilisers, suspending agents,fillers, glidants, compression aids, binders or tablet-disintegratingagents; it can also be an encapsulating material. In powders, thevehicle is a finely divided solid that is in admixture with the finelydivided active polypeptide or derivative. In tablets, the activepolypeptide or derivative is mixed with a vehicle having the necessarycompression properties in suitable proportions and compacted in theshape and size desired. The powders and tablets preferably contain up to99% of the active polypeptide or derivative. Suitable solid vehiclesinclude, for example, calcium phosphate, magnesium stearate, talc,sugars, lactose, dextrin, starch, gelatin, cellulose,polyvinyipyrrolidine, low melting waxes and ion exchange resins.

Liquid vehicles are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active polypeptide orderivative can be dissolved or suspended in a pharmataceuticallyacceptable liquid vehicle such as water, an organic solvent, a mixtureof both or pharmaceutically acceptable oils or fats. The liquid vehiclecan contain other suitable table pharmaceutical additives such assolubilisers, emulsifiirs, buffers, preservatives, sweeteners,flavouring agents, suspending agents, thickening agents, colours,viscosity regulators, stabilizers or osmo-regulators. Suitable examplesof liquid vehicles for oral and parenteral administration include water(partially eomaiuiug additives as above, e.g. cellulose derivatives,preferably sodium earboxymethyl cellulose solution), alcohols(including, iuonohydric alcohols and polyhydric alcohols, e.g. glycols)and these derivatives, and oils (e.g. fractionated coconut oil andarachis oil). For parenteral administration, the vehicle cast also be anoily ester such as ethyl oleate and isopropyl myristate. Sterile liquidvehicles are useful in sterile liquid form compositions for parenteraladministration. The liquid vehicle for pressurized compositions can behalogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by for example, intramuscular, intrathecal,epidural, intraperitoneal, intravenous and particularly subcutaneous,intracerebral or intracerebroventricular injection. The polypeptide orderivative may be prepared as a sterile solid composition, that may bedissolved or suspended at the time of administration using sterilewater, saline, or other appropriate sterile injectable medium. Vehiclesare intended to include necessary and inert binders, suspending agents,lubricants, flavouraols, sweeteners, preservatives, dyes, and coatings.

Polypeptides or derivatives according to the invention can beadministered orally in the form of a sterile solution or suspensioncontaining other solutes or suspending agents (for example, enoughsaline or glucose to make the solution isotonic), bile salts, acacia,gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitoland its anhydrides copolymerized with ethylene oxide) and the like.

The polypeptides can also be administered orally either in liquid orsolid composition form. Compositions suitable for oral administrationinclude solid forms, such as pills, capsules, granules, tablets, andpowders, and liquid forms, such as solutions, syrups, elixirs, andsuspensions. Forms useful for parenteral administration include sterilesolutions, emulsions, and suspensions.

The polypeptides or derivatives may be used to treat any mammal, forexample, human, livestock, pets, to prevent infection from occurring.

By way of example, polypeptides or derivatives in accordance with theinvention may be used to prevent candida infections. When this is thecase the medicament may be formulated as a cream and it may be used inthe form of a pessary.

By way of further example, polypeptides or derivatives in accordancewith the invention may be used to prevent infections of “athlete'sfeet”. When this is the case the medicament may be formulated as a creamand it may be applied to the affected area of the skin.

By way of further example, polypeptides or derivatives in accordancewith the invention may be used to prevent or treat malaria. When used toprevent infection then it is preferred that a therapeutically effectiveamount of the polypeptide (e.g. about 10 mg of the polypeptide) isadministered stored to the subject's skin in a suitable composition, forexample, either by a cream, lotion or aerosol. It will be appreciatedthat methods exist, which aim to either prevent or treat malaria in man.One such treatment method (or regime) consists of intravenouslyadministering the compound Quinine into a patient. The loading dose isapproximately 15 mg/kg quinine base in about 10 ml/kg normal saline or5% dextrose, and is injected into the subject over a period of about 4hours. Subsequent maintenance doses of about 8.3 mg/kg quinine base arethen infused into the patient or subject over 4 hours, and every 8hours, until an administration of oral quinine is possible. In patientswho require more than 72 hours of intravenous treatment, the dose may bereduced to about 5.6 mg base per kg given every 8 hours. The inventor ofthe present invention believes that peptides in accordance with theinvention may be used either on their own, or in conjunction with, or asa supplement to, existing malaria treatment regimes. For example,therapeutically effective amounts (e.g about 10 mg) of the peptideaccording to the invention may be added to the solution of quinone to beinjected in to the subject. The peptide according to the invention mayor may not be administered orally. However, it is preferred that thepeptides are not administered orally.

In accordance with a fourth aspect of the invention, there is provided amethod of preventing and/or treating a fungal and/or protist infection,comprising administering to a subject in need of such treatment atherapeutically effective amount of a polypeptide, derivative, oranalogue or nucleic acid according to the invention.

The method according to the fourth aspect of the invention may employany medicament and any use discussed in connection with the first,second and third aspects of the invention.

It will be appreciated that fungal and protist infections can also be aproblem in plants important to horticulture and/or agriculture.Therefore the formulations of peptides discussed above may be adaptedfor application to plants. Thus according to a fifth aspect of theinvention there is provided a method of preventing or treating a fungaland/or protist infection of a plants comprising applying a polypeptideas defined in the first, second or third aspects of the invention to aplant in need of such treatment.

The polypeptides as defined above may be used as anti-microbial agentsfor spraying on crops and the like. For instance the peptides may alsobe used to treat fungal infections of plant species, including fungalcontamination of cereal crops, fungal contamination of stored grain,potato blight and downy mildew of grapes.

Examples of fungal species, which cause agriculturally important fungalinfections (for example in plants), and which may be treated by thepeptides according to the invention may include preferred Ascomycetes,which may be independently selected from a group consistingof:—Erisyphe; Puccinia; Leptoshaeria; Thanatephorus; Pyricularia;Phytopthora; Plasmopara; Alternaria; Gulgnardia; Pseudocerocosporella;Venturia; Monolinia; and Ustilago. In addition, further examples ofagriculturally important fungi include Botryotinia spp; and Cochilobusspp; and most preferably, Magnaporthe spp. A preferred Magnaporthe spp.includes M. grisea.

The polypeptides may be used to treat any plant species suffering from afungal or protist infection. A skilled person will appreciate thatprecise formulation and dosage for agricultural or horticultural usewill depend on the peptide used, the type of plant treated, the size ofplant treated and also the scale of treat required (for instance manyacres or a single plant may be required to be treated). In general anamount of the polypeptide effective for treating a single potato plantis about 0.01-100 mgs. It is more preferred that about 10 mg of thepolypeptide administered either directly onto the plant or to its rootsin a suitable formulation, for example, either by a liquid or spray.

Polypeptides used according to the fifth aspect of the invention may beadded to existing formulations used to treat plants, such as pesticides,weedkillers etc. For example, Ridomil Gold MZ may be applied to foliageof potato plants to control late blight caused by Phytophthorainfestans. Early in the season, 1.2 Kg of Ridomil Gold MZ may be appliedto the crop per acre. Preventative treatments are begun when conditionsare favourable for disease (i.e preferably, before infection). Up tothree applications may be made, at 14 day intervals. The inventorbelieves that such regimes for preventing potato blight may besuppiernented and surprisingly improved by incorporating peptidesaccording to the invention into the Ridomil Gold MZ solution.

The inventor has realised that the polypeptides according to theinvention may also be put to a number of other antimicrobial uses(whether in a clinical context or otherwise). For instance, in additionto administering the polypeptides to a patient, animal or plant, theymay be used to coat surfaces and objects to prevent or treat fungaland/or a protist contamination.

Therefore, in a sixth aspect there is provided a method of preventingand/or treating a fungal and/or protist contamination comprising coatingan object or a surface in need thereof with an amount of a polypeptideaccording to the first, second or third aspect of the invention, that iseffective for killing or preventing growth of fungi and/or protists.

It will be appreciated that the polypeptide may be particularly usefulfor coating surfaces or objects that are required to be aseptic. Asdiscussed above, many of the polypeptides have the advantage that theyare antifungal, antiprotist, and in addition, also antiviral andantibacterial. Accordingly, the polypeptide will have a very broadanti-microbial effect across several kingdoms. Furthermore, as discussedin more detail below, the polypeptides are able to adhere to surfacesand are thereby effective for longer periods of time.

The polypeptides may be used to coat any object or device which is usedin a biological or medical situation, such as a medical device, and forwhich it may be important to prevent a fungal or protist contaminationthat may lead to any infection in a patient. Examples of medical devicesthat may be coated according to the sixth aspect of the inventioninclude lenses, contact lenses, catheters, stents, wound healingdressings, contraceptives, surgical implants and replacement joints.

The polypeptides are particularly useful for coating biomaterials andobjects and devices made therefrom. Fungal or protistcontamination/infection of biomaterials can be particularly problematicbecause the fungus or protist may use such material as a substrate forgrowth. Biomaterials (e.g. collagens and other biological polymers) maybe used to surface artificial joints. Alternatively certain implants maysubstantially comprise such biomaterials.

The polypeptides may be used to coat surfaces in environments that arerequired to he aseptic. For instance the polyperides may be used inmedical environments. The polypeptides may be used to keep hospitalwards clean. They may be used to clean surfaces of equipment (e.g.operating tables) in operating theatres as well as theatre walls andfloors. The inventors believe the polypeptides will be useful to improvesterility in general.

The polypeptides may be formulated into solutions for cleaning objectsand surfaces. For instance, they may be a routine constituent ofphysiological solutions (for example as a constituent of physiologicalsaline).

Example 3 illustrates how well polypeptides in accordance with theinvention adhere to contact lenses. Hence, the peptides according to theinvention are very useful as they have been shown to adhere strongly toan article or surface used in a biological scenario.

It will be appreciated that the above list of objects and surfaces towhich the polypeptides according to the invention may be applied is notexhaustive. Hence, the polypeptides maybe administered to any surface,which is prone to a fungal or protist contamination, for example,kitchen and bathroom surfaces and products, such as a toilet seat, orthe toilet itself.

In a preferred embodiment, the polypeptides may be included in salinesolution used to store contact lenses.

Preferred polypeptides according to the invention are highly positivelycharged. This makes them particularly suited for coating surfaces andobjects to prevent growth, of broad categories of fungi and protists.Example 3 and FIGS. 3 and 4, clearly illustrate how well thepolypeptides in accordance with the invention adhere to a range ofdifferent surfaces, ie, glass (cover slips), glass previously coatedwith the biomalerlal Poly(lactide-co-glycolide) (PLGA), and contactlenses.

Preferably, coating of the object or surface may be carried out bypreparing an aqueous solution at an appropriate pH and temperature forthe said polypeptides according to the invention. The object or surfaceis exposed to the said solution for sufficient time to allowimmobilisation or absorption of a suitable quantity of the polypeptidesto the surface thereof or to allow sufficient time to kill the fungus orprotist.

In a preferred embodiment of the sixth aspect of the invention, asufficiently concentrated solution of a polypeptide according to theinvention is prepared, and contacted with the object to be coated for asuitable period of time. The skilled technician will appreciate how tomake a polypeptide solution of the required concentration, as this willdepend on the particular polypeptide being used and the fungus orprotist to be treated, and the surface being coated. For examples theobject may be inserted in the solution (e.g. comprising about 40 μM ofthe polypeptide) and left for about 15 minutes at about 20° C. Followingexposure to the polypeptide, the object may be washed, for example, in asuitable buffer, such as, PBS. It may be required to leave the object inthe wash buffer overnight. Following washing, the polypeptide has thenadhered to the object, and the object, coated with the protectivepolypeptide, is ready for use.

According to a seventh aspect of the invention there is provided acontact lens at least partially coated with a polypeptide according tothe first, second or third aspects of the invention. The polypeptideapplied to the surface of the contact lens prevents fungal and/orprotist contamination occurring that can results in infections occurringin the eye of the user.

In one embodiment, the lens may be a one day disposable lens (i.e. wornfor one day and then disposed of), in which case, fungal or protistcontamination is obviated before the lens is used and also when removedfrom its package. Accordingly, the lens my be pre-treated with thepolypeptide and/or may be packaged, in a solution containing thepolypeptide. The lens coated with the polypeptide reduces the likelihoodof a fungal infection in the user than may occur while the contact lensis being worn.

Alternatively, a lens may be repeatedly worn on a dally basis forseveral months or years, but taken out and washed and stored in solutionover night. When this is the case a polypeptide coating on the lens(before first use) and/or preferably use of the polypeptides in lenswash solutions, will significantly reduce the likelihood of a fungal orprotist infection of the user occurring while the lens is being worn, orthe lens being contaminated while it is being stored and washedovernight.

In another embodiment, the lens may be an extended wear lens, which isconstantly worn in the eye for extended periods of time, for example,more than one day, several days, a week or even a month or more. Usersof such contact lenses have a high risk of developing a fungal orprotist infection. Hence, in this case, the polypeptide may be used tocoat the lens before it is first-used. Use of such a coated lens willgreatly reduce the likelihood of a fungal or protist infection occurringwhile the lens is being worn for such extended periods of time.

In a preferred embodiment, a contact lens is coated with a polypeptideaccording to the invention, and where appropriate, stored and/or washedin a solution comprising the polypeptide.

It will be appreciated that agents that increase the activity ofpolypeptides or derivatives or analogues according to the invention maybe used to “indirectly” increase the activity of such polypeptides,derivatives or analogues. Thus, according to an eighth aspect of theinvention, there is provided an agent capable of increasing thebiological activity of a polypeptide, derivative or analogue accordingto the first, second or third, aspect of the invention for use as amedicament for treating a fungal and/or protist infection.

Agents capable of increasing the biological activity of polypeptides,derivatives or analogues according to the invention may achieve theireffect by a number of means. For instance, such agents may increase theexpression of such polypeptides, derivatives or analogues. Alternatively(or in addition), such agents may increase the half-life ofpolypeptides, derivatives or analogues according to the invention in abiological system, for example, by decreasing turnover of thepolypeptides, derivatives or analogues.

The inventor has further established that several polypeptides or agentsaccording to the invention may be combined and used to prevent or treata broad range of fungal or protist infections/contaminations (as well asviral and bacterial infections/contaminations). For example, it may bepreferred to treat a fungal or protist infection/contamination with acombination of polypeptides according to any one of the first, second,or third aspects, such as a polypeptide independently selected from agroup consisting of MU4, MU7, MU10 or MU114. However, it will beappreciated that different combinations of polypeptides can be used toprevent or treat different fungal or protist infections.

Furthermore, the polypeptide and agents according to the invention maybe used to minimise, prevent or treat fungal or protist contamination orgrowth, by use as, or in conjunction with, a preservative. Hence, thepolypeptides and agents may be used as a preservative in foodstuffs. Inaddition, the polypeptides and agents may be used to minimise or preventfungal or protist growth in cultures, for example, in tissue culturework, either to supplement, or to replace antibiotics and otherantifungal/antiprotist agents. In addition, the polypeptides may be usedas selective agents as a diagnostic agent, for example, for fungal orprotist growth in culture media. For example, a first polypeptide may beadded to media, which is particularly active against a first fungus, anda second polypeptide may be added to the media, which is particularlyactive against a second fungus. A similar method could be used fordiagnosing protista.

The polypeptides, analogues, or derivatives of the invention representproducts that may advantageously be expressed by biological cells.Therefore the present invention also provides, in a ninth aspect, anucleic acid sequence encoding a polypeptide, derivative or analogueaccording to the first, second or third aspects of the invention.

Preferred nucleic acids according to the ninth aspect of the inventionmay be selected from the group consisting of: SEQ ID No.56(cttcgtaaacttcgtaaacgtcttctt), SEQ ID No.5 (cgtcttactcgtaaacgtggtcttaaa), SEQ ID No.58(cttcgtaaacgtcttcttcttcgtaaacttcgtaaacgtcttctt), SEQ ID No. 59(caatctactgaagaacttcgtgttcgtcttgctagtcatcttcgtaaacttcgtaaacgtcttctt),SEQ ID No.60(cttcgtgttcgtcttgctagtcatcttcgtaaacttcgtaaacgtcttcttcgtgatgctgatgatcttcaaaaacgtcttgctgtttatcttcgtgttcgtcttgctagtcatcttcgtaaacttcgtaaacgtcttcttcgtgatgctgatgatcttcaaaaacgtcttgctgtttat), SEQ ID No.61(cttcgtaaacttcgtaaacgtcttcttcttcgtaaacttcgtaaacgtcttctt), SEQ ID No.62(tggcgtaaatggcgtaaacgttggtggtggcgtaaatggcgtaaacgttggtgg), SEQ ID No.63(tggcgtaaatggcgtaaacgttggtggcgtaaatggcgtaaacgttgg), SEQ ID No.64(tggcgttaaat ggcgtaaacgttggtggcttcgtaaacttcgtaaacgtcttctt), SEQ ID No.65(tatcgtaaatatcgtaaacg ttattattatcgtaaatatcgtaaacgttattat), SEQ ID No.66(cttcgtaaacttcgtaaacgtcttcgtaaacttcgtaaacgt), SEQ ID No.67(cgtcttactcgtaaacgtgg tcttaaacgtcttactcgtaaacgtggtcttaaa), SEQ ID No.68(cgtactcgtaaacgtggtcgtcgtactcgt aaacgtggtcgt), SEQ ID No.69(cttcgtaaacgtaaacgtcttcttcgtaaacgtaaacgtctt), SEQ ID No.70(cttcgtaaacgtaaacgtcttcgtaaacttcgtaaacgtaaacgtcttcgtaaa), SEQ ID No.71(tggcgttggcgtaaacgttggcgtaaatggcgttggcgtaaacgttggcgtaaa), SEQ ID No.72(MU4) (tggcgtaaatggcgtaaacgttggtggtggcgtaaatggcgtaaacgttggtgg), SEQ IDNo.73 (MU7) (tttcgtaaatttcgtaaacgtttttttttttcgtaaatttcgtaaacgttttttt),SEQ ID No.74 (MU10)(ttacgtaaattacgtaaacgtttattattacgtaaattacgtaaacgtttatta), and SEQ IDNo.75 (MU114) (tggcgtaaatggcgtaaacgtttattattacgtaaattacgtaaacgtttatta).

Preferred nucleic acids further include those corresponding DNAmolecules encoding any preferred polypeptides according to theinvention.

It will be appreciated that, due to redundancy in the genetic code, anucleic acid sequence in accordance with the invention may vary from thenaturally occurring sequence (e.g. in the ApoB or ApoE genes) providinga codon encodes a polypeptide, derivative or analogue thereof inaccordance with the first, second or third aspect of the invention.

It will be appreciated that polypeptides, derivatives and analoguesaccording to the invention represent favourable agents to headministered by techniques involving cellular expression, of nucleicacid sequences encoding such molecules. Such methods of cellularexpression are particularly suitable for medical use in which thetherapeutic effects of the polypeptides, derivatives and analogues arerequired over a prolonged period.

Thus according to a tenth aspect of the present invention there isprovided a nucleic acid sequence according to the previous aspect of theinvention for use as a medicament.

According to an eleventh aspect, there is provided use of the nucleicacid, for the preparation of medicament for treating a fungal and/orprotist infection.

The nucleic acid may preferably be an isolated or purified nucleic acidsequence. The nucleic acid sequence may preferably be a DNA sequence.

The nucleic acid sequence may further comprise elements capable ofcontrolling and/or enhancing its expression. The nucleic acid moleculemay be contained within a suitable vector to form a recombinant vector.The vector may for example be a plasmid, cosmid or phage. Suchrecombinant vectors are highly useful as delivery systems fortransforming cells with the nucleic acid molecule.

Recombinant vectors may also include other junctional elements. Forinstance, recombiaaaat vectors can be designed such that the vector willautonomously replicate in the cell. In this case elements that inducenucleic acid replication may be required in the recombinant vector.Alternatively, the recombinant vector may be designed such that thevector and recombinant nucleic acid molecule integrates into the genomeof a cell. In this case nucleic acid sequences, which favour targetedintegration (e.g. by homologous recombination) are desirable.Recombinant vectors may also comprise DNA coding for genes that may beused as selectable markers in the cloning process.

The recombinant vector may also further comprise a promoter or regulatorto control expression of the gene as required.

The nucleic acid molecule may (but not necessarily) be one, whichbecomes incorporated in the DNA of cells of the subject being treated.Undifferentiated cells may be stably transformed leading to theproduction of genetically modified daughter cells (in which caseregulation of expression in the subject may be required e.g. withspecific transcription factors or gene activators). Alternatively, thedelivery system may be designed to favour unstable or transienttransformation of differentiated cells in the subject being treated.When this is the case, regulation of expression may be less importantbecause expression of the DNA molecule will stop when the transformedcells die or stop expressing the protein (ideally when the requiredtherapeutic effect has been achieved).

A delivery system may provide the nucleic acid molecule to the subjectwithout it being incorporated in a vector. For instance, the nucleicacid molecule may be incorporated within a liposome or virus particle.Alternatively a “naked” nucleic acid molecule may be inserted into asubject's cells by a suitable means, e.g, direct endocytotic uptake.

The nucleic acid molecule may be transferred to the cells of a subjectto be treated by transaction, infection, microinjection, cell fusion,protoplast fusion or ballistic bombardment. For example, transfer may beby ballistic transfection with coated gold particles, liposomescontaining the nucleic acid molecule, viral vectors (e.g. adenovirus)and means of providing direct nucleic acid uptake (e.g., endocytosis) byapplication of the nucleic acid molecule directly.

Embodiments of the invention, will now be further described, by way ofexample only, with reference to the following Example and figures inwhich:

FIG. 1 shows typical mass spectrometry data and illustrates that thepeptide was >95% purity;

FIG. 2 shows typical HPLC data and illustrates that the peptide was >95%purity;

FIG. 3 illustrates Johnson and Johnson Acuvue contact lenses, which hadbeen treated for 15 min with 40 μM GIN1p (which had been synthesisedwith the addition of a cysteine residue having a fluorescent tag), thenwashed 4 times, including an overnight soak in 25 ml PBS as discussed inExample 3;

FIG. 4 illustrates glass cover slips (BG), or cover slips previouslycoated with the biomaterial Poly(lactide-co-glycolide) (PLGA), which hadbeen treated for 15 min with 40 μM GIN1p (which had been synthesizedwith a fluorescent tag), then washed 4 times, including an overnightsoak in 25 ml PBS as discussed in Example 3; and

FIG. 5 illustrates inhibition of hepatocyte invasion, by plasmodium sppmediated by peptides according to the invention fo: (A) cells incubated,with Plasmodium and peptide; and (B) cells mcdhated with Plasmodium,washed, and then peptide added as discussed in Example 5.

EXAMPLES

The inventor carried out a number of experiments to investigate theantifungal and also antiprotist activity of polypeptides according tothe invention. The activity of the polypeptides was tested against anumber of different fungi (Examples 1 and 4) and protists (Example 2 and5). In addition, the inventor investigated the ability of thepolypeptides to adhere to a variety of surfaces, for example, contactlenses, glass and surfaces coated with the biomaterial “PLGA.” (Example3) and thereby prevent fungal or protist contamination.

Peptides

Peptides (moulding polypeptides according to the invention) wereobtained in lyophilised form from a commercial supplier (AltaBioscience,University of Birmingham), and were produced at 5 micromole scale. Theskilled technician will know the standard techniques, which areavailable for synthesisiog peptides, once an amino acid sequence hasbeen made available. N-terminals were protected by addition of an acetylgroup, and the C-terminals were protected by addition of an amide group.Small quantities of peptide were weighed in sterile Eppendorf tubes,before addition of sufficient PBSA to produce a 0.4 mM stock solution,which was frozen at −85° C. in aliquots.

Molecular weight of peptides was confirmed by laser desorption massspectrometry using a Finnigan LASERMAT 2000 MALDI-time of flight massanalyzer or a Scientific Analysis Group MALDI-TOF mass spectrometer.HPLC purification of peptides was performed using a Vydac analytical C-4reverse phase column, using 0.1% TFA and 0.1% TFA/80% acetonitrile assolvents, or for some peptides an ACE C18 Reverse Phase column, using0.05% TEA and 60% acetonitrile as solvents. Typical mass spectrometrydata and high performance liquid chromatography (HPLC) traces(purity>95%) for peptide GIN1p are shown in FIGS. 1 and 2.

Example 1 Experiment to Test the Antifungal Activity of Peptides

Four compounds were supplied as an aqueous solution, 400 μM. Serialdilutions of each test peptide were prepared in addition to a control,which was a conventional antifungal drug, Amphoteracin B. Amphteracin Bis obtained from Invitrogen or Melford laboratories.

The peptides under test were:

(i) MU4- (SEQ ID No. 9) WRKWRKRWWWRKWRKRWW; (ii) MU10- (SEQ ID NO. 8)LRKLRKRLLLRKLRKRLL; and (iii) MU114- (SEQ ID NO. 26) WRKWRKRLLLRKLRKRLL.

Initial testing of the peptides was carried out against the followingorganisms:

-   1) Aspergillus fumigatus AF293 (culture collection strain NCPF7367)-   2) Candida albicans 6862 (clinical isolate)-   3) Fusarium spp 5889 (clinical isolate)-   4) Fusarium spp 6507 (clinical isolate)-   5) Aspergillus terreus (clinical isolate)-   6) Aspergillus niger (clinical isolate)-   7) Staphylococcus aureus (Oxford strain, which is a standard    reference strain used in MIC testing)

The bacterium S. aureus was tested with each of the peptides as acontrol. A suspension of each organism in an appropriate growth mediumwas added to the dilutions of the peptides in order to giveconcentration ranges of 40 μM to 0.04 μM for each of the peptides, and64-0.025 μg/ml for amphotericin B. Fungal strains were tested in RPMImedia. RPMI is standard Roswell Park Memorial Institute medium (Morton,H.C., (1970) A survey of Commercially Available Tissue Culture Media. InVitro 6: 89-108).

S. aureus was tested in Isoseositest broth. Isosentitest agar is astandard MIC medium used by many hospital labs for bacterial MICs usinga disc diffusion method. The reference standard is Oxoid CM471. Alsoavailable in broth form, CM473, for the bacterial broth dilution assaymethod. NCCLS ref M7-A4. Both the NCCLS methods quoted are the standardmethods.

The final inoculum for S. aureus was 5×10⁴ cfu/ml. The final inoculumfor C. albicans was 2×10³ cfu/ml. The final inoculum for Aspergillusfumigatus and for Fusarium spp was 2×10⁴ cfu/ml.

Candida and S. aureus plates were read after 24 hrs incubation and theAspergillus and Fusarium strains read after 48 hrs. The MIC was taken asthe lowest drag or compound concentration that caused >80% reduction ingrowth compared with a drug free control.

TABLE 2 Antifungal effect of peptides MU_10 MU_114 Amphotericin MU_4MICs MICs MIC in μg/ml MICs in μM in μM in μM C. albicans 6862 0.610 >40 >40 Aspergillus 0.6 2.5 >40 >40 fumigatus Fusarium spp 5889 2.52.5 10 5 Fusarium spp 6507 1.25 10 >40 40 Aspergillus terreus1.25 >40 >40 >40 Aspergillus niger 0.15 1.25 >40 20 Staph aureus >405 >40 40 (Oxford)

From Table 2, it will be seen that peptide MU4 is the most active of thepeptides tested having activity against all four fungal species tested,and also surprisingly, against S. aureus (a gram positive bacterium).Peptide MU4 is also active against Aspergillus niger. However, MU4 showslower activity against Aspergillus terreus, which is known to beresistant to various antifungal drugs.

MU10 and MU114 also showed antifungal activity, albeit to a lesserextend than MU4. However, MU10 and MU114 showed antifungal activityagainst Fusarium 5889. The activity of the peptides is surprising inthat they are more active against Fusarium spp than against Aspergillusspp.

Conclusion

Using standard antifungal testing methods, the peptides according to theinvention are shown to have antifungal activity. In addition,surprisingly, some antibacterial activity is also present. Peptide MU4is particularly potent against Aspergillus fumilgatus, Aspergillus nigerand Fusarium spp. The other peptides (MU10 and MU114) are less active,but still, show antifungal activity.

Example 2 Experiment to Test the Antiprotist Activity of Peptides

The test compounds were screened against the trophozoites of clinicalisolates of Acanthamoeba spp. using a microtitre plate assay procedure.The organisms were Acanthamoeba polyphaga. (see for details on thestrain and methodology used: Hughes, R. & Kilvingtom S. (2001). Acomparison of hydrogen peroxide contact lens disinfection systems andsolutions against Acanthamoeba polyphaga. Antimicrob. Agents Chemother.45; 2038-2043.) Acanthamoeba polyphaga Ros strain was used throughoutthe study. The strain was originally isolated from an republished caseof acanthamoeba keratitis in the United Kingdom in 1994.

Use of the microtitre plate assay enabled the determination of theminimum amoebacidal trophozoite concentration (MATC), which is theminimum amoebacidal trophozoite concentration (μg/ml)=lowestconcentration killing the trophozoite challenge (1×10-4) and also theminimum inhibitory trophozoite concentration (MITC), which is theminimum inhibitory trophozoite concentration (μg/ml)=lowestconcentration stopping the trophozoites from dividing or killing about50% of population. The values may then be used to select drugconcentrations for use in time-kill experiments to study the kinetics ofAcanthamoeba killing.

The peptides under test were:

(i) MU4- (SEQ ID No. 9) WRKWRKRWWWRKWRKRWW; (ii) MU7- (SEQ ID No. 15)FRKFRKRFFFRKFRKRFF. (iii) MU10- (SEQ ID NO. 8) LRKLRKRLLLRKLRKRLL; and(iv) MU114- (SEQ ID NO. 26) WRKWRKRLLLRKLRKRLL.

TABLE 3 Antiprotist Effects of Peptides Values in μM Drug MITC MATC MU 420.8 41.7 MU 7 NR 93.1 MU 10 NR 207.1 MU 114 NR 98.7 MITC = minimuminhibitory trophozoite concentration (μM) MATC = minimum amoebacidaltrophozoite concentration (μM) NR = Not recorded as a MITC was notobserved

Conclusion

Referring to Table 3, it can be seen that MU4 showed the greatestantiprotist activity having the lowest MITC and MATC values by killingAcanthamoeba polyphaga. MU 7 and MU114 showed good activity againstAcanthamoeba polyphaga as they showed an MATC of 93.1 and 98.8,respectively. Finally, MU10 also showed antiprotist activity with anMATC of207.1.

Example 3 Coating of Medical Devices with Antifungal/AntiprotistPolypeptides

Polypeptides according to the invention may be used to coat medicaldevices prone to contamination with fungi or protists. Examples of suchmedical devices include lenses, catheters, stents, wound healingdressings and contraceptive devices. The polypeptides may also beapplied to surfaces in medical environments, including surfaces ofequipment for use in operating theatres. The inventors of the inventionhave shown that the polypeptides according to the invention adhere tocontact lenses, glass and to surfaces coated with the biomaterial“PLGA”.

Coating a surface can be carried out by preparing a concentrated aqueousSolution of a polypeptide (for example 200 μM) at an appropriate pH (forexample pH7.4) for the specific polypeptide. A surface is then exposedto the aqueous solution at a suitable temperature (for example 3° C.)for sufficient time (for example 2 hours) to allow immobilisation orabsorption of a suitable quantity of the polypeptide to the surfacethereof.

To test whether the peptides according to the invention could beimmobilised on a biomaterial or other surfaces, the inventors obtained afluorescently labelled form of the GIN1p (Advanced Biomedical, Oldham,UK). A 40 μM stock solution of this labelled polypeptide was prepared inPBS, and 250 μl allquots were inserted in to the wells of a 24-wellmicroplate. The inventors then placed several materials into thesepeptide solutions, these being: (i) Johnson and Johnson Acovne contactlenses; (ii) bare glass coverslips; or (iii) coverslips previouslycoated with the biomaterial Poly(lactide-co-glycolide) (PLGA: coatedslides provided by Prof Jian Lu, Department of Physics, University ofManchester).

After incubation at 20° C. for 15 mm in the peptide solutions, thematerials were removed, and then washed by placing in 1 ml PBS. Thematerials were then examined by fluorescence microscopy (using anOlympus IX70 inverting microsoope fitted with a Cinema 35002v2 filterset), and the results were recorded by photography. The materials werethen washed two more times in 1 ml PBS, and then finally left to soakovernight at 37° C. in 25 ml PBS. The level of fluorescence was observedand recorded after each wash, again by microscopic observation andphotography as described above.

Referring to FIG. 3, there are shown Johnson and Johnson Acuvue contactlenses, which had been treated for 15 min with 40 μM GIN1p (i.e. MU10),(which had been synthesized with a fluorescent tag), then washed 4times, including an overnight soak in 25 ml PBS. FIG. 3( a) shows anuntreated lens and a GIN1p-treated lens (after 4 washes), underillumination with white light, or GFP fluorescence, using a Olympus IX70microscope. Hence even after repeated washing, the lens retains asignificant quantity of peptide, such that fluorescence is visible evenby eye as shown in FIG. 3( b). The images were captured using a CanonEOS300D digital camera, using ISO1600 film setting, and with a 0.3 sexposure time for fluorescent images.

Referring to FIG. 4, there are shown glass cover slips (BG), or coverslips previously coated with the biomaterial Poly(lactide-co-glyeclide)(PLGA), which had been treated for 15 min with 40 μM GIN1p (which hadbeen synthesised with a fluorescent tag), then washed 4 times, includingan overnight soak in 25 ml PBS. The level, of fluorescence was observedusing an Olympus IX70 microscope for samples after each wash (W1, W2, W3and W4), Hence, it can be seen that the level of fluorescence did notdecrease noticeably after any of the washes, suggesting that thepolypeptide adheres firmly to a range of surfaces. The images werecaptured using a Canon EOS300D digital camera, with a 5 s exposure timeusing ISO1600 film setting.

Accordingly, FIGS. 3 and 4 show that all three types of materialappeared to retain similar levels of GIN1p despite extensive washing,suggesting the polypeptide is suitable for coating various surfaces (asshown in FIG. 1 a, and FIG. 4). In particular, the contact lenses werefound to absorb significant quantities of the polypeptide (presumablydue to their large surface area), such that fluorescence was clearlyvisible to the naked eye, even after the fourth overnight wash as shownin FIG. 3 b.

Conclusions

From Table 2, it will be seen that the polypeptides in accordance withthe present invention show antifungal activity against at least one, ifnot two, and if not three, of the six different fungi trains evaluated.In particular, MU 4, MU 10, and MU 114 are particularly effective. Oneof the most active peptides is MU 4, which is active against C. albicans6862, Aspergillus fumigaius, Fusarium spp 5889, Fusarsium spp 6507,Aspergillus termus and Aspergillus niger.

From Table 3, it will be seen that polypeptides in accordance with thepresent invention also show antiprotist activity against Acantamoebapolyphaga. In particular, MU 4, MU 7, MU 10) and MU 114 are particularlyeffective.

It is worth noting that MU 4 was surprisingly active against fungi andprotists.

Each of these polypeptides in accordance with the invention are derivedfrom a Heparan Sulphate Proteoglycan (HSPG) receptor binding region ofapolipoprotein E. In addition, each polypeptide is a tandem repeat, andcomprise two RKR motifs. The data illustrates the surprising propertythat tandem repeats in accordance with the invention are effectiveantifungal and antiprotist agents.

Example 4 Antifungal Efficacy Against a Number of Fungi

The inventor expanded the work conducted in Example 1 by testing anexpanded library of peptides derived from either apolipoprotein B orapolipoprotein E in order to further evaluate polypeptides in accordancewith the invention. These experiments were conducted using the followingfungal species:

Fusarmm solani 1 (FS1);

Fusarium soiam 2 (FS2);

Fusarium solani 3 (FS3);

Fusarium saltan 4 (FS4);

Aspergillus fumigatus 293(AF); and

Candida albicans (CA),

FS1-FS4 were obtained from Bristol PHLS (British Public HealthLaboratory Service UK). Aspergillus fumigatus 293 is publicly availableas culture collection strain NCPF7367 (National Collection of Pathogenicfungi Public Health Laboratory, Mycological Reference Laboratory MyrtleRoad, Kangsdown, Bristol BS2 8EL) The Candida albicans was a clinicalisolate obtained by standard means.

The methods used to determine the IC₅₀ values for each peptide were asdescribed in the Example 1. An IC₅₀ of less or about 40 μM was taken tohave good antifungal activity

Table 4 provides data for peptides that are derived from apoE.

TABLE 4 MIC values (>80% reduction) against 6 fungal species of Peptides derived from apoEPeptide SEQ. ID. No. Sequence FS1 FS2 FS3 FS4 AF CA MU_1 SEQ ID NO. 76ERKERKREEERKERKREE >40 >40 >40 >40 >40 >40 (GIN 6) MU_2 SEQ ID NO. 77ARKARKRAAARKARKRAA >40 >40 >40 >40 >40 >40 (GIN 39) MU_3 SEQ ID NO. 78DRKDRKRDDDRKDRKRDD >40 >40 >40 >40 >40 >40 MU_4 SEQ ID NO. 9WRKWRKRWWWRKWRKRWW 0.3 0.3 0.3 0.3 1.25 2.5 (GIN 7) MU_5 SEQ ID NO. 32MRKMRKRMMMRKMRKRMM 10 20 20 40 >40 >40 (GIN 40) MU_6 SEQ ID NO. 12YRKYRKRYYYRKYRKRYY 10 5 5 20 >40 >40 (GIN 41) MU_7 SEQ ID NO. 15FRKFRKRFFFRKFRKRFF 5 2.5 5 20 >40 >40 MU_8 SEQ ID NO. 33IRKIRKRIIIRKIRKRII 40 20 20 40 >40 >40 MU_9 SEQ ID NO. 79QRKQRKRQQQRKQRKRQQ >40 >40 >40 >40 >40 >40 MU_10 SEQ ID NO. 8LRKLRKRLLLRKLRKRLL 2.5 5 2.5 5 >40 >40 (GIN 1p) MU_11 SEQ ID NO. 80NRKNRKRNNNRKNRKRNN >40 >40 >40 >40 >40 >40 MU_12 SEQ ID NO. 30CRKCRKRCCCRKCRKRCC 2.5 5 10 5 40 >40 MU_13 SEQ ID NO. 81SRKSRKRSSSRKSRKRSS >40 >40 >40 >40 >40 >40 MU_14 SEQ ID NO. 82VRKVRKRVVVRKVRKRVV >40 >40 >40 >40 >40 >40 MU_15 SEQ ID NO. 83TRKTRKRTTTRKTRKRTT >40 >40 >40 >40 >40 >40 MU_16 SEQ ID NO. 31RRKRRKRRRRRKRRKRRR 1.25 1.25 1.25 2.5 40 10 MU_17 SEQ ID NO. 84GRKGRKRGGGRKGRGRGG >40 >40 >40 >40 >40 >40 MU_18 SEQ ID NO. 85KRKKRKRKKKRKKRKRKK 2.5 10 2.5 >40 >40 >40 MU_19 SEQ ID NO. 34HRKHRKRHHHRKHRKRHH 20 >40 40 10 >40 >40 MU_20 SEQ ID NO. 86PRKPRKRPPPRKPRKRPP >40 >40 >40 >40 >40 >40 GIN 34 SEQ ID NO. 11WRKWRKRWWLRKLRKRLL 1.25 0.6 1.25 2.5 40 10 MU_45 SEQ ID NO. 87 WRKWRKRWW40 40 40 >40 >40 >40 MU_82 SEQ ID NO. 21 LRKLRKRLLRLRKLRKRLLR 2.5 2.52.5 10 >40 >40 MU_112 SEQ ID NO. 24 LRKLRKRLLLRKLRKRWW 2.5 2.5 2.55 >40 >40 Amphotericin B (antifungal positive control) 0.15 1.25 0.30.15 0.04

In Table 4 it will be appreciated, that MU1-MU20 correspond to tandemrepeat of peptides based on apoE₁₄₁₋₁₄₉, (MU10) in which each L residueis substituted with another amino acid (MU1-MU9 and MU11-MU20).Polypeptides in accordance with the present invention exhibited goodantifungal activity with IC₅₀ values<40 μM against at least four of thefnngi tested. MU 4, MU 6, MU 7, MU10, MU12 MU16 and MU 18 areparticularly effective antifungal peptides. The most preferred peptideMU4 has activity comparable with amphotericin B. Table 4 illustratesthat a range of peptides based upon substituted tandem repeats ofpeptides based on apoE₁₄₁₋₁₄₉, which fall outside the definition of thepolypeptides according to the invention, are ineffective relative topeptides according to the invention.

It will be seen that the preferred polypeptide MU 4 (a tandem repeat ofWRKWRKRWW) has good antifungal activity whereas the monomer on which itis based MU45 is ineffective. This illustrates that tandem repeats ofApoE₁₄₁₋₁₄₉ and derivatives thereof (as defined by herein) aresurprisingly effective antifungal agents.

Table 5 presents data for peptides that are derived from apoB.

TABLE 5 MIC values for peptides derived from apolipoprotein B SEQ. ID.Sequence FS1 FS2 FS3 FS4 AF CA Peptide No. MU_25 SEQ IDWRWRRRWRKWRWRRRWRK 5 5 10 20 10 >40 NO. 88 MU_26 SEQ IDWRWKKKWRKWRWKKKWRK 5 5 5 10 >40 >40 NO. 89 MU_27 SEQ IDWRWRKRWRKWRWRKRWRK 0.6 0.6 0.6 1.25 5 20 (GIN 33) NO. 55 MU_28 SEQ IDRRWRKRWRKWRWRKRWRK 0.6 0.6 1.25 2.5 10 >40 NO. 42 Amphotericin B 0.151.25 0.3 0.15 0.04 (antifungal positive control)

From Table 5, it will be seen that the peptides MU27 and MU28, which,are peptides in accordance with the present invention, show goodantifungal activity against at least five of the different fungalstrains evaluated.

It will be appreciated that MU25 and MU26 have reduced activity. Thesepeptides closely resemble peptides MU27 and MU28 according to theinvention but have had the RKR motifs modified or changed.

Example 5 Antimalarial Efficacy of Peptides According to the Invention

Experiments were conducted to illustrate that peptides according to theinvention are effective at preventing infection of cultured hepatocytesby plasmodium sporozites and thereby demonstrate that the peptides haveefficacy for preventing and treating malaria.

Methods

Preparation of Plasmodiaum Sporozoites: 3-5 day-old Anopheles stephensimosquitoes were fed on anesthetized P. berghei (NK65)-infected SwissWebster mice which had been checked by blood smear for the abundance ofgametocyte-stage parasites. Salivary gland sporozoites were harvested ondays 18 to 21 post-infective blood meal. The mosquitoes were rinsed in70% ethanol and washed in Dulbecco's Modified Eagle Medium (DMEM) beforesalivary gland dissection. The glands were gently ground, spun at 80×gfor 3 min to remove mosquito debris and sporozoites counted in ahemocytometer.

Sporozoite-Development Assay: Hepa 1-6 cells (ATCC CRL-1830, AmericanType Culture Collection., Manassas, Va.), a mouse hepatoma cell linepermissive for P. betghei sporozoite development were seeded (8×10⁴cells/well) in Lab-Tek permanox chamber slides (Nalgene Nunc Corp.,Napervllle, Ill.) and grown until confluent. On the day of theexperiment, sporozoites were dissected from mosquitoes and pre-incubatedwith Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Carlsbad,Calif.) with 1% BSA alone or with the indicated polypeptide at 50 μg/mlfor 1 hr at 28° C. and plated on cells in the continued presence of theinhibitor in DMEM containing 10% fetal calf serum (complete medium).After 1 hr at 37° C., the medium containing unattached sporozoites andpolypeptide was removed and replaced with complete medium. 40 hourslater the cells were fixed with cold methanol and exoerythrocytic stages(EEFs) were stained with mAb 2136 (Tsuji, M., D. Mattel, R. S.Nussenzweig, D. Eichinger, and F. Zavala. 1994. Demonstration ofheat-shock protein 70 in the sporozoite stage of malaria parasites.Parasitology Research 80:16-21.) followed by anti-mouse immunoglobulinconjugated to FITC. The number of EEFs in each well were counted with a40× objective on a Nikon fluorescent microscope. In other experiments,Hepa1-6 cells were pretreated with the polypeptides at 50 μg/ml incomplete medium for 1 hour, washed and then sporozoites were added incomplete medium without polypeptide and the assay was continued asoutlined above.

Results

FIG. 5 shows that both MU10 and MU4 inhibit entry of P berghei into Hep1-6 cells. In additional experiments in which cells were treated withthese peptides, washed using cell culture media, before final challengewith P berghei, MU4 retained its ability to block P berghei entry,suggesting that this compound may bind the surface of these cellsirreversibly, with this then later preventing entry of the parasite.This rodent model is widely used to study the entry of Plasmodium intoliver; activity as demonstrated here almost certainly means that entryof Plasmodiumi falciparum (and other Plasmodium species) into liverwould be similarly prevented.

These data illustrate that peptides according to the invention may beused in the treatment of malaria and other protist infectious.

1-24. (canceled)
 25. A method of preventing, reducing the likelihood, or treating a fungal or protist infection in a subject, comprising: administering to a subject in need of said prevention, reduction of likelihood or treatment an amount of a polypeptide that is effective for killing or preventing or reducing the likelihood of growth of fungi or protists; and wherein the polypeptide comprises a 14-29 amino acid polypeptide derived from a Heparan Sulphate Proteoglycan (HSPG) receptor binding region of an apolipoprotein and comprises at least two RKR motifs.
 26. The method according to claim 25, wherein the polypeptide is derived from a Heparan Sulphate Proteoglycan (HSPG) receptor binding region of apolipoprotein B or apolipoprotein E.
 27. The method according to claim 25, wherein the polypeptide is derived from a Heparan Sulphate Proteoglycan (HSPG) receptor binding region of apolipoprotein B LDL receptor binding domain cluster B or apolipoprotein E LDL receptor binding domain cluster B.
 28. The method according to claim 25, wherein the polypeptide comprises a tandem dimer repeat of SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; or derivatives thereof wherein at least one amino acid, other than RKR motifs, is replaced by an Arginine (R), Tyrosine (Y), Methionine (M), Isoleucine (I), Phenylalanine (F), Tryptophan (W), Cysteine (C) or a derivative thereof.
 29. The method according to claim 25, wherein at least one amino acid, other than RKR motifs, is replaced by an Arginine (R), Phenylalanine (F) or Tryptophan (W) or a derivative thereof.
 30. The method according to claim 25, wherein the polypeptide is of formula (I): abcRKRxyza′b′c′RKRx′y′z′  (I) wherein a & a′=is independently selected from Arginine (R): Tyrosine (Y); Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W); Leucine (L); Lysine (K); Histidine (H); Cysteine (C) or is deleted; b & b′=is independently selected from Arginine (R): Tyrosine (Y); Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W); Leucine (L); Lysine (K); Cysteine (C) or is deleted; c & c′=is independently selected from Arginine (R): Tyrosine (Y); Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W); Leucine (L); Lysine (K); Histidine (H); Threonine (T); Cysteine (C); or is deleted; x & x′=is independently selected from either Arginine (R): Tyrosine (Y); Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W); Leucine (L); Lysine (K); Histidine (H); Glycine (G); Cysteine (C) or is deleted; y & y′=is independently selected from Arginine (R): Tyrosine (Y); Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W); Leucine (L); Lysine (K); Histidine (H); Cysteine (C) or is deleted; z & z′=is independently selected from Arginine (R): Tyrosine (Y); Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W); Leucine (L); Lysine (K); Histidine (H); Cysteine (C) or is deleted; wherein the polypeptide is 14-18 amino acids in length; and wherein optionally an amino acid that is independently selected from Arginine (R): Tyrosine (Y); Methionine (M); Isoleucine (I); Phenylalanine (F); Tryptophan (W); Leucine (L); Lysine (K)and Histidine (H) may be added before the amino acid at position “a”.
 31. The method according to claim 25, wherein the polypeptide comprises a repeat of apoE₁₄₁₋₁₄₉ (SEQ ID NO: 5) or a variant thereof.
 32. The method according to claim 31, wherein the polypeptide comprises at least two substitutions independently selected from Tryptophan (W); Arginine (R); Lysine (K); Tyrosine (Y) or Phenylalanine (F) substitutions.
 33. The method according to claim 31, wherein the polypeptide comprises the amino acid sequences as defined by any one of SEQ ID NOs: 8-34.
 34. The method according to claim 25, wherein the polypeptide comprises a repeat of apoB₃₃₅₉₋₃₃₆₇ (SEQ ID NO: 6) or a variant thereof.
 35. The method according to claim 34, wherein the polypeptide comprises the amino acid sequences as defined by any one of SEQ ID NOs: 36-55.
 36. The method according to claim 25, for preventing, reducing the likelihood, or treating a an Ascomycete fungal infection.
 37. The method according to claim 25, for preventing, reducing the likelihood, or treating an Onygenales fungal infection.
 38. The method according to claim 37, wherein the fungus is a Epidermophyton spp., Microsporum spp. or Trichophyton spp.
 39. The method according to claim 37, wherein the fungus is a Candida spp
 40. The method according to claim 25, wherein the peptide is formulated for topical application.
 41. The method according to claim 25, wherein the polypeptide has an IC₅₀ of 75 μM or less.
 42. The method according to claim 25, wherein the polypeptide is used to prevent, reduce the likelihood, or treat athletes foot.
 43. A method of reducing the likelihood of, or treating a fungal or protist contamination of a non-living object or surface, comprising: applying to the object or surface an amount of a 14-29 amino acid polypeptide that is effective for killing or inhibiting growth of fungi or protists, wherein the polypeptide is derived from a Heparan Sulphate Proteoglycan (HSPG) receptor binding region of an apolipoprotein.
 44. A composition, comprising: a polypeptide comprising a 14-29 amino acid polypeptide derived from a Heparan Sulphate Proteoglycan (HSPG) receptor binding region of an apolipoprotein and comprises at least two RKR motifs; and a pharmaceutically acceptable carrier. 